AIR™ System User Manual

CellRaft AIR™ System

Automated Isolation and Retrieval

Single cell screening, isolation and retrieval using CellRaft™ Technology

User Manual CellRaft AIR™ System User Manual

Table of Contents

Notices ...... 4 Contact Information ...... 4 Document Conventions ...... 5 Revision History ...... 6 1. Intended Use Statement ...... 7 2. Warnings and Precautions ...... 8 2.1. Warnings ...... 9 2.2. Hazards and Precautions ...... 10 2.3. Electromagnetic Compatibility ...... 12 2.4. User Safety ...... 12 3. Warranty ...... 13 4. Introduction ...... 14 4.1. AIR™ Instrument ...... 15 4.2. Core Features ...... 15 4.3. Theory of Operation...... 15 4.3.1. The CytoSort Array ...... 15 4.3.2. CellRaft Selection through Imaging ...... 16 4.3.3. Fluorescence Excitation and Emission Bands of the CellRaft AIR System ...... 16 4.3.4. Needle-Based Release and Magnetic Collection of the CellRaft ...... 17 5. Installation ...... 18 5.1. Package Contents ...... 19 5.2. AIR Instrument Specifications ...... 19 5.3. Unpack and Inspect the AIR™ Instrument ...... 20 5.4. Select Appropriate Location ...... 20 5.5. Connect Power Supply and System Computer ...... 21 5.6. Computer and Portable Solid State Drive Compatibility ...... 22 5.7. Software Overview...... 23 5.8. Installing the AIR System Software ...... 23 6. System Operation ...... 25 6.1. Power on the AIR™ Instrument, Computer, Mouse and Keyboard ...... 26 6.2. Key Operational Components ...... 26 6.2.1. Power Switch ...... 26 6.2.2. Instrument Door ...... 26 6.2.3. AIR™ Instrument Fan and Air Intake ...... 26 6.2.4. AIR™ System Microscope Stage ...... 27 6.2.5. CytoSort Array Adapter Plate...... 27 6.2.6. 96-well culture plate compatibility ...... 28 6.2.7. PCR Tube Compatibility ...... 29 6.3. Experimental Design with the AIR System ...... 31 6.4. Hardware and Software Initialization ...... 33 6.4.1. Software Start-up and System Initialization ...... 33

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6.4.2. Creating a new database record for a CytoSort Array ...... 33 6.4.3. Loading an existing database record for a CytoSort Array ...... 35 6.5. AIR System Software Home Screens ...... 37 6.5.1. AIR software module Home screen ...... 37 6.5.2. Off the AIR software module Home screen ...... 40 6.6. Selecting CellRafts for Isolation using User Navigation and Selection Mode ...... 44 6.6.1. Entering User Navigation and Selection mode ...... 44 6.6.2. Real-Time Imaging in User Navigation and Selection mode ...... 45 6.6.3. CellRaft Selection for Isolation in User Navigation and Selection mode ...... 49 6.7. Acquiring a Full Array Scan on a CytoSort Array ...... 51 6.7.1. Entering Full Array Scan mode ...... 51 6.7.2. Imaging Setup for a Full Array Scan ...... 52 6.7.3. Executing the Full Array Scan in the Array Scanning screen...... 55 6.8. User Review and Selection Mode of Full Array Scan Data ...... 56 6.8.1. Entering User Review and Selection mode ...... 56 6.8.2. Image Review in User Review and Selection mode ...... 57 6.8.3. CellRaft Selection for Isolation in User Review and Selection mode ...... 60 6.9. Cytometric Image Analysis Mode of Full Array Scan Data...... 62 6.9.1. Entering Cytometric Image Analysis mode ...... 63 6.9.2. CellRaft Gating in Cytometric Image Analysis mode ...... 63 6.9.3. Marker Gating in Cytometric Image Analysis mode ...... 70 6.9.4. CellRaft Selection for Isolation in Cytometric Image Analysis mode ...... 75 6.10. Tracking CellRafts using the CellRaft Organizer ...... 79 6.10.1. Entering the CellRaft Organizer screen ...... 79 6.10.2. CellRaft Organizer functionality ...... 80 6.11. Release and Transfer of CellRafts Selected for Isolation ...... 85 6.11.1. Initiating CellRaft Isolation ...... 85 6.11.2. Executing CellRaft Isolation ...... 87 6.11.3. Confirming Isolation of CellRafts in the Collection Plate ...... 89 6.12. Import a list of CellRaft Addresses for Isolation ...... 90 6.13. Experimental Report and Images ...... 92 6.13.1. Microsoft Excel report ...... 92 6.13.2. Exported Images ...... 94 7. Troubleshooting ...... 95 7.1. Overview ...... 96 7.2. Testing Power Connection ...... 96 7.3. Testing Computer Connection ...... 96 7.4. High number of CellRaft isolation failures ...... 97 8. Appendix 1 – Labware Compatibility ...... 98 8.1. Validated flat-bottom 96-well culture plates ...... 99 8.2. Validated PCR tubes ...... 99 8.3. Validated PCR plates ...... 99 9. Appendix 2 – Fluorescent Dye Compatibility ...... 100 9.1. Fluorescent Dyes and Stains Compatible with the AIR System ...... 101

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Notices

Cell Microsystems, Inc. 2 Davis Dr. PO Box 13169 Research Triangle Park North Carolina, 27709

All Rights Reserved © 2019, Cell Microsystems, Inc. No part of this publication may be reproduced, transcribed, or transmitted in any form, or by any means electronic or mechanical, including photocopying and recording, for any purpose other than the purchaser’s use without written permission of Cell Microsystems, Inc.

Contact Information

Cell Microsystems, Inc. 2 Davis. Dr. P.O. Box 13169 Research Triangle Park North Carolina, 27709 USA

Internet: http://www.cellmicrosystems.com Phone: 252-285-9842

To purchase CytoSort Arrays or other Cell Microsystems products, contact [email protected].

For technical questions and other support, contact [email protected].

For general information about Cell Microsystems, contact [email protected].

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Document Conventions

This icon calls attention to important safety notes.

Warning! A warning indicates the potential for bodily harm and tells you how to avoid the problem.

Caution A caution indicates potential damage to the instrument and tells you how to avoid the problem.

Note: Bold text is primarily used for emphasis.

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Revision History

Document Number: PD-017

Rev Date Changes 00 25 JAN 2019 Initial Release 01 23 APR 2019 Addition of Cytometric CellRaft Gating modes (on top of single-cell detection of a nuclear stain): o Gate based on fluorescence intensity thresholds o Gate based on number of cells as estimated from brightfield images Addition of Cytometric Marker Gating metrics, based on: o Number of cells on CellRaft estimated from brightfield images o Confluence of colony growth on CellRaft estimated from brightfield images o Delta in new and existing metrics between two full array scans Implementation of Cytometric Marker Gate “promotion” for a gate population to be available during Plate Mapping, included in the list of Marker Gates on the Home screen, and included in the Marker Gate filter list in the CellRaft Organizer Ability to assign “tags” to CellRafts in form of star rating and thumbs up or thumbs down to track CellRafts of interest over time or to exclude CellRafts from analyses Addition of CellRaft Organizer screen with the ability to: o Display a table of CellRafts in the CytoSort Array o Filter the CellRaft table based on star rating, thumbs up/down rating, Cytometric Marker Gates, collection plate, color group, isolation status, time since modified or viewed o View images of CellRafts selected from the table o Map collection plates from the CellRaft table or modify existing plate maps Minor edits to instruction screens 02 05 JUN 2019 Addition of Access Wand button to AIR module Home screen to allow user to easily remove wand after CellRaft isolation Addition of unskirted and semi-skirted 96-well PCR plate compatibility 03 04 SEP 2020 Addition of compatibility with 100m and 200m CytoSort HexaQuad Arrays Revision of interface tools to search for and load a database record Addition of reservoir within a multi-reservoir array as a filter option within the CellRaft Organizer

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1. Intended Use Statement

The CellRaft Automated Isolation and Retrieval (AIR™) System is a computer-attached, bench-top instrument employing the proprietary CellRaft™ Technology for optical imaging, screening and subsequent isolation of cells from the CytoSort™ Array.

For Research Use Only. Not for use in diagnostic procedures.

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2. Warnings and Precautions

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2.1. Warnings

❖ For Research Use Only. Not for use in diagnostic procedures.

❖ Care should be taken when loading and unloading the instrument with CytoSort Arrays or collection well plates to not spill liquid into the instrument which could damage the underlying electrical components or cause a safety hazard.

❖ Use only the power cord provided with the instrument.

❖ Use only the provided USB 3.0 cords for attaching peripherals to the AIR™ Instrument (i.e. computer).

❖ Use of the instrument outside of the specified environmental conditions may degrade the safety features of this instrument.

❖ Physiological samples of all types from any source should be treated as biohazardous according to your organization’s approved procedures.

❖ If the instrument is used in a manner not specified by Cell Microsystems, Inc. equipment protections against injury may be impaired.

❖ Operate the instrument on a flat surface away from excessive humidity and moisture.

❖ Performance can be adversely affected by extraneous contaminants such as dust, dirt, adhesive, etc. on the CytoSort Array. Make sure CytoSort Arrays are clean and free of debris before use.

❖ When operated in a safe environment according to the instructions in this document, there are no known hazards associated with the use of this instrument. However, the operator should be aware of certain conditions that could result in injury. See Hazards and Precautions for more information.

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2.2. Hazards and Precautions

Hazards Warning! Power Rating. The instrument’s power cord must be connected to a power receptacle that provides voltage and current within the specified rating for the system. Use of an incompatible power receptacle may produce electrical shock and fire hazards.

Warning! Electrical Grounding. Never use a two-prong plug adapter to connect primary power to the external power supply. Use of a two-prong adapter disconnects the utility ground, creating a severe shock hazard. Always connect the power cord directly to a three-prong receptacle with a functional ground.

Warning Internal Voltage. Always turn off the power switch and unplug the power supply before cleaning the outer surface of the instrument. Failure to do so may produce an electrical shock hazard.

Warning! Liquids. Avoid spilling liquids on the instrument; fluid seepage into internal components creates a potential shock hazard. Wipe up all spills immediately. Do not operate instrument if internal components have been exposed to fluid.

Warning! Potential Biohazards. Some assays or specimens may pose a biohazard. Adequate safety precautions should be taken as outlined in your organization’s standard operating procedures.

Warning! Unspecified Use. Failure to operate the instrument according to the guidelines set forth in this document could result in a hazardous condition.

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Precautions Caution: Service. The AIR™ Instrument should be serviced by Cell Microsystems’ authorized service personnel only. Any user-performed service of the instrument should be restricted to those steps described in the “Troubleshooting” section of this document.

Caution: Environmental Conditions. Do not expose the system to temperature extremes. For proper operation, ambient temperatures should remain between 15 and 30°C. Performance may be adversely affected if temperatures fluctuate above or below this range. Storage temperature limits are broader (-20 to +50°C).

Caution: Instrument Cleaning. Clean the instrument with approved solutions only. Do not expose any part of the instrument to the recommended cleaning solution for more than 10 minutes. Prolonged contact with cleaning solution may damage the instrument surfaces. Be certain to rinse and thoroughly wipe all surfaces. Should flammable liquids (e.g. isopropanol or ethanol) be used to clean the system, care should be taken to avoid open flames.

Caution: Operating Voltage. Only use the power cable supplied with the instrument. Operate the instrument only within the range of line voltages listed.

Caution: Shipping Fixtures. Remove all shipping fixtures before powering the instrument on. See section 5.3 for instructions. These fixtures must be reinstalled when repackaging the instrument, for shipment or otherwise.

Caution: Warranty. Failure to follow preventative maintenance protocols may void the warranty.

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2.3. Electromagnetic Compatibility

The CellRaft AIR™ System has been tested by TUV SUD America and has been found to be compliant to the following standards: Emissions: FCC Part 15B, ICES-003 FCC Part 15.109(a):2017 & ICES-003 Issue 6:2017 FCC Part 15.107(a):2017 & ICES-003 Issue 6:2017

2.4. User Safety

The CellRaft AIR™ System has been tested by TUV SUD America and has been found to conform to IEC 61010-1, 3rd Edition (2010) Safety Standards. Certifications are represented on the product label, an example of which is shown here in Figure 1.

Figure 1: Product label displaying electrical ratings and product certifications.

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3. Warranty

Products sold by Cell Microsystems have been designed and tested to perform according to the published specifications. Cell Microsystems warrants that the products it manufactures and sells will conform to those specifications and be free from defects in workmanship and materials for a period of twelve (12) months form the date of shipment. Defective products will be repaired or replaced, at Cell Microsystems option, during this period provided the purchaser has used the products under conditions of normal and proper use, but not for damage caused by the purchaser.

THE FOREGOING WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY MADE BY CELL MICROSYSTEMS. CELL MICROSYSTEMS MAKES NO OTHER WARRANTY OR GUARANTEE OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING WITHOUT LIMITATION IMPLIED WARRANTIES OF FITNESS FOR A PARTICULAR PURPOSE OR MERCHANTABILITY. CELL MICROSYSTEMS’ TOTAL LIABILITY AND PURCHASERS EXCLUSIVE REMEDY FOR ANY CLAIM OR LIABILITY ASSOCIATED WITH THE PRODUCTS SOLD, WHETHER BASED IN TORT, CONTRACT, STRICT LIABILITY OR ANY OTHER LEGAL THEORY IS EXPRESSLY LIMITED TO, AT CELL MICROSYSTEMS OPTION, REPLACEMENT, REPAIR, OR REWORK, AS APPLICABLE, OF NONCONFORMING PRODUCT OR PAYMENT IN AN AMOUNT NOT TO EXCEED, IN THE AGGREGATE, THE PURCHASE PRICE OF THE SPECIFIC PRODUCT FOR WHICH DAMAGES ARE CLAIMED. IN NO EVENT SHALL CELL MICROSYSTEMS BE LIABLE FOR ANY OTHER DAMAGES, LOSSES OR EXPENSES, INCLUDING, WITHOUT LIMITATION, INDIRECT, INCIDENTAL, PUNITIVE, CONSEQUENTIAL, EXEMPLARY, OR SPECIAL DAMAGES.

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4. Introduction

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4.1. AIR™ Instrument

The AIR™ Instrument is a computer-attached bench-top instrument. It is intended to be used with microwell arrays manufactured by Cell Microsystems called CytoSort™ Arrays. Cells are cultured on CytoSort Arrays allowing imaging-based sorting and isolation using the automated AIR™ System. The graphical user interface allows scanning of the array to image the cells and automated detection of single cell candidates for isolation using image processing algorithms.

4.2. Core Features

• Automated three-channel fluorescence and brightfield imaging, factory configured with 10X magnification • Microneedle release of individual CellRafts (i.e. a single microwell position on the CytoSort Array) for subsequent collection using a motorized magnetic wand • Collection of CellRafts isolated from the CytoSort Array microwells into individual wells or tubes of 96-well flat-bottom culture plates and 0.2mL PCR tube strips and plates • SBS-format microscope stage for loading the CytoSort Array and multi-well collection plate • Operated using the graphical user interface software installed on an attached computer

4.3. Theory of Operation

4.3.1. The CytoSort Array

The CytoSort™ Array comprises elastomeric (polydimethylsiloxane or PDMS Well Array PDMS) microwell arrays, with each well of the array filled with an individually releasable polystyrene element called a CellRaft (Figure 2, bottom). Cells are seeded on the CytoSort Array and settle by gravity into the microwells in a random distribution. Refer to the CytoSort Array Polystyrene CellRaft™ User Manual for cell seeding densities to achieve efficient distribution of single cells in single microwells (Figure 2, middle). Figure 2: The CytoSort Array comprises a PDMS well array The array is housed in a petri dish-sized cassette (Figure 2, top) with walls and polystyrene CellRafts surrounding the array to create a reservoir of media, buffer or other where cells are seeded and reagents shared by all microwells and their resident cells. Refer to the imaged. The entire CellRaft is collected during isolation.

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CytoSort Array Manual for recommended fluid compositions and volumes within each reservoir to maximize CellRaft isolation efficiency for a given CytoSort Array type.

The CytoSort Arrays can be surface coated similar to any plastic cell culture consumable with extracellular matrix proteins or non-specific biological adhesives to render them compatible with non- adherent cell types. Refer to the CytoSort Array Manual for additional details regarding array preparation and coating instructions.

NOTE: The CytoSort Arrays are single-use consumables. At the time of manufacturing a coating is applied to the surface of the array to allow liquids to flow easily into the microwells of the array without forming large image-occluding bubbles. This coating cannot be re-applied for a second use and therefore the arrays are single use only. Re-use of CytoSort Arrays will significantly decrease the efficiency of cell seeding and adherence as well as release and retrieval of CellRafts.

4.3.2. CellRaft Selection through Imaging

The AIR™ System allows brightfield and three-color fluorescence imaging of cells seeded and cultured on the CytoSort Array. Supported imaging modalities include: • Full Array Scan (section 6.7) – automated scans of every CellRaft within the array • User Navigation and Selection (section 6.6) – real-time imaging of user-selected fields of view within the CytoSort Array and individual selection of CellRafts for isolation. Both modes provide full user control of the camera imaging parameters (exposure, binning, and gain).

Subsequent to a Full Array Scan, CellRafts containing single cells or colonies of interest can be identified based on their brightfield and fluorescence properties by user review of the images (User Review and Selection, section 6.8) or using the AIR System software’s automated image processing pipeline (Cytometric Image Analysis, section 6.9). If the imaging capabilities of the AIR System are not conducive to CellRaft selection for a given experiment, the user can compile a list of array locations of interest using a separate microscope and import the list into the software (section 6.12) to still use the AIR System to perform automated CellRaft isolation into a collection plate.

4.3.3. Fluorescence Excitation and Emission Bands of the CellRaft AIR System

The CellRaft AIR System provides three fluorescent imaging channels in addition to brightfield. When selecting fluorophores for experiments, consult Table 1 for the standard AIR System fluorescence excitation and emission specifications. More detailed information to determine compatibility of specific fluorescent dyes and stains is included in Appendix 2 – Channel Representative Dyes Excitation Emission Fluorescent Dye Compatibility. In Blue Hoechst, SYTO 41 378-401 nm 412-453 nm the event that the fluorescence Green FITC, Alexa488 460-490 nm 497-548 nm imaging channels were customized Red Texas-Red 559-591 nm 602-805 nm during manufacturing, consult the Table 1: Excitation and emission specifications for the three fluorescence imaging channels enabled by the AIR™ System (standard configuration).

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imaging specifications provided Imaging Nominal Imaging with the custom AIR System. Dye Concentration Exposure Channel SYTO® 13 Green 5 μM 2 ms Green NOTE: Within Cytometric Image SYTO® 17 Red 5 μM 15 ms Red Analysis mode, the AIR System SYTO® 41 Blue 5 M 50 ms Blue software is capable of Hoechst 33342 1.6 μM (1 μg/mL) 20 ms Blue automatically identifying which Table 2: List of example dyes which can be used to stain nuclei in each of the CellRafts within the Full Array standard AIR System fluorescent imaging channels. Note that any of the Scan contain a single cell. Single- fluorescent channels can be used to count the cells during Cytometric Analysis. cell detection can be accomplished based on brightfield images or by applying a nuclear fluorescent dye to all cells in the experiment. Details on brightfield-based cell counting can be found in section 6.9.

For fluorescence-based single-cell detection, stains exhibiting strong nuclear localization promote a more accurate count of cells on each CellRafts. Any of the three fluorescence channels can be used for this automated analysis feature. The use of a nuclear stain to serve as the “Cell Counting Channel” and details for setting the parameters for automated single cell detection can be found in section 6.9. See Table 2 for suggestions of vital dyes which provide reliably strong nuclear signal that have been validated for use on the AIR System by Cell Microsystems.

4.3.4. Needle-Based Release and Magnetic Collection of the CellRaft

Once CellRafts are selected for isolation, the AIR™ System employs a proprietary release, collection, and deposit strategy for CellRaft isolation into a collection plate. The AIR™ System will move the CytoSort Array over a motorized microneedle. Once in position the needle is actuated upward, penetrating the resealable PDMS microwell floor and pressuring the hard polystyrene CellRaft out of the microwell. Once free, the CellRaft is collected by actuating a magnetic wand over the CellRaft location. The system will detect successful release from the microwell and collection by the magnetic wand and repeat the process multiple times until the CellRaft is collected. The wand then deposits the CellRaft into an individual well or tube of a collection plate (e.g. 96-well culture or PCR plates, PCR tube strips). Once all selected CellRafts have been isolated in the wells or tubes of the collection plate, the CytoSort Array and collection plate can be retrieved by the user.

If the CellRaft is not successfully released and collected from the microwell array, the system will report the error to avoid downstream analytical issues with suspected empty wells or doublets (should the CellRaft be collected in subsequent operations). At present, the AIR System does not: 1) allow multiple CellRafts to be isolated into a single well or tube of the collection plate, nor 2) confirm successful deposit of CellRafts into the collection plate.

NOTE: It is recommended to visually inspect the tubes or wells of the collection plate to confirm collection of CellRafts. Refer to section 6.11.3 for more details.

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5. Installation

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5.1. Package Contents

• AIR™ System Instrument, including: o Power cord (3 meters) o Two (2) high-performance USB 3.0 cables (3 meters) • AIR™ System Accessory Kit: o CytoSort Array Adapter Plate o PCR Tube Adapter Plate o Two (2) Standard Collection Wands o Two (2) PCR Tube Collection Wands • USB 3.0 portable solid-state drive including: o AIR™ System software o Off the AIR software installer o AIR™ System User Manual o CytoSort Array User Manual o AIR™ System QuickStart Guide o DB Browser for SQLite installer o AIR System database table structure o Example query scripts for use in DB Browser for SQLite software • (optional) AIR™ System All-In-One Computer including: o Power cord (1.5 meters) o Wireless keyboard and mouse

5.2. AIR Instrument Specifications

• Power Supply Input voltage: 100 – 240 VAC, 50/60Hz, 570W • Environmental Operating Temperature: +15°C to +30°C Operating Humidity: 20 – 80% non-condensing Operating Altitude: 0 – 7500 ft above mean sea level Storage Temperature: -20°C to +50°C

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5.3. Unpack and Inspect the AIR™ Instrument

Important! Save all packing materials.

Upon receipt, inspect the packaging, instrument and other parts and accessories for shipping damage. If you suspect that the instrument has been mishandled in shipping, notify the carrier and Cell Microsystems. Keep the instrument and all packing materials undisturbed for the carrier’s inspection.

The CellRaft AIR System instrument is shipped with stabilizing brackets to protect critical internal components during shipping. Removal of these brackets is required during installation prior to system operation and must be performed by trained Cell Microsystems personnel. As such, do not attempt to operate the AIR System prior to the installation date scheduled with your Cell Microsystems sales or technical support representative.

If moving the system after installation more than approximately 25 feet, the system should be re-crated and moved using a dolly or other moving device. If the crate is moved by truck or other vehicle, it is recommended to re-install the stabilizing brackets prior to re-crating. Contact Cell Microsystems for detailed instructions or to schedule a service visit.

5.4. Select Appropriate Location

The CellRaft AIR™ System should be installed on a level, stable surface where ambient temperatures between 10°C and 30°C can be maintained.

The instrument is sensitive to environmental conditions. Avoid the following:

• Excessive Humidity: Condensation directly on the electronics may present a shock or fire hazard. The specified relative humidity range of the analyzer is 20 to 80% (non-condensing). • Excessive Ambient Light: Bright sunlight or fluorescent light may degrade the quality of images taken while cells are on the instrument. Operate away from excessive light, and never operate with the instrument door open. • Dust: Excessive dust and debris on the CytoSort Array surface can create imaging artifacts which impact the success of automated single cell identification and/or cause contamination of the cell culture. Operate in a clean environment.

NOTE: The CellRaft AIR System can be operated in a horizontal laminar flow hood with interior dimensions of at least 31”W x 24”H x 24”D. For more information, contact Cell Microsystems sales or technical support.

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5.5. Connect Power Supply and System Computer

Warning! Power Rating. The instrument’s power cord must be connected to a power receptacle that provides voltage and current within the specified rating for the system. Use of an incompatible power receptacle may produce electrical shock and fire hazards.

Perform these steps to connect the power supply (Figure 3): 1. Connect the AIR System power cord to the back of the instrument. 2. Plug the power cord into an appropriate power receptacle.

Perform these steps to connect the AIR™ Instrument to the system computer (Figure 3): 1. Connect the USB 3.0 cords to the back of the AIR™ Instrument. 2. Connect the other ends of the USB cords to two USB 3.0 ports (often coded with blue flanges and marked with an “SS” designation) on the rear of the system computer. Figure 3: Power and USB ports on the AIR™ Instrument

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5.6. Computer and Portable Solid State Drive Compatibility

An AIR™ System instrument control computer should be chosen in consultation with Cell Microsystems based on the following compatibility criteria:

- Windows 10 or Windows 7 (SP1) - Intel I7, Intel I9, AMD FX or AMD Ryzen series microprocessor (4 Core minimum) - Minimum 8GB dynamic RAM - Minimum 256GB Solid State Hard Drive - Minimum two (2) USB 3.1 data ports for machine operation - Recommended additional USB 3.1 Gen 2 data port for portable data applications - Recommended minimum 1920 x 1080 display

Installation of the Off the AIR software module should be limited to desktop computers or laptops that meet the following compatibility criteria:

- Windows 10 or Windows 7 (SP1) - Intel I7, Intel I9, AMD FX or AMD Ryzen series microprocessor (4 Core minimum) - Minimum 8GB dynamic RAM - Recommended USB 3.1 Gen 2 data port for portable data applications - Recommended minimum 1920 x 1080 display

To facilitate transfer of AIR System data from the AIR™ System instrument control computer to an external computer, it is recommended to purchase a portable flash drive capable of the fastest possible data transfer rate that matches the capability of the internal hard drive and USB ports on the AIR System control computer (e.g. a solid-state drive (SSD) with USB 3.1 version 2). Each complete scan of a CytoSort Array may take up to 10GB of storage, so a minimum drive capacity of 0.5TB is recommended.

Recommend device families include: • Western Digital “My Passport” portable SSDs • Samsung Portable SSD T5 • SanDisk Extreme Portable SSD Please use care when ordering, as the companies above use similar names for their internal hard drives and portable drives. Make sure the drive is SSD, portable, and USB 3.1 capable. Contact Cell Microsystems for specific options. For recent AIR System purchases, a compatible SSD is provided with the instrument.

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5.7. Software Overview

The AIR System software is split into two software modules – AIR and Off the AIR. The AIR software module controls all system functionality that requires the AIR System hardware, such as CytoSort Array imaging and CellRaft isolation into collection plates. The Off the AIR software module contains software functionality that does not require the AIR System hardware, such as image analysis and CellRaft selection for isolation. As such, the Off the AIR software can be installed on any computer with a Windows operating system (Windows 7 SP1 or later) and USB 3.1 Gen 2 data port (recommended). The user can analyze imaging data and select CellRafts containing single cells or colonies of interest without monopolizing the AIR instrument and while cells seeded on the CytoSort Array are recovering in the incubator.

The AIR System Software uses a SQLite database to manage the data generated by the AIR System. Images, collection plate maps, and isolation history associated with a given CytoSort Array is consolidated into a single database record which can be stored on the computer attached to the AIR System instrument or on a portable solid-state drive (SSD) such as the one provided with the instrument. Such flexibility allows easy transport of all AIR System data back and forth between the AIR System computer and the user’s office computer.

If the CellRaft AIR System was purchased with an accompanying computer, both software modules come pre-loaded on the computer. If not, the software modules can instead be installed on any compatible computer (see section 6.1 for compatibility requirements) according to the installation instructions in section 5.8. As indicated above, the Off the AIR module can also be installed on any compatible computer for image analysis and CellRaft selection.

The following sections will provide step-by-step instructions for installing the AIR System Software. Sections 6.4 – 6.13 will provide navigation guidance for all functions of the software user interface.

5.8. Installing the AIR System Software

If the CellRaft AIR System was purchased with an accompanying computer, the AIR System computer will arrive with all AIR System software pre-installed. The Desktop icon labeled AIR refers to the AIR instrument control software and is used to communicate with the AIR System instrument during imaging and isolation processes. The Desktop icon labeled Off the AIR is used to analyze data within a database record acquired from the instrument.

To install on a user-supplied AIR System computer for instrument control: 1. Create a CMS directory within the C:\ directory of the user-supplied AIR System control computer.

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2. Plug the Cell Microsystems portable solid-state drive (SSD) provided with the instrument into a USB C or USB A port on the AIR System computer using the appropriate cable supplied with the portable SSD. 3. Navigate to the CMS folder on the portable SSD, copy the AIR folder, and paste it into the C:\CMS folder on the AIR System computer. All executables and libraries needed for operation on the machine should be included in this folder. 4. Shortcuts for the two executables (AIR.exe and OffTheAIR.exe) should be provided in the AIR folder. Copy these shortcuts and place them on the Desktop of the AIR System computer.

To install the Off the AIR software for image analysis and plate mapping on a separate computer: NOTE: Do NOT install the Off the AIR software on the AIR System computer if provided by Cell Microsystems as both software modules are already configured for connection to the AIR System. 1. Plug the Cell Microsystems portable solid-state drive (SSD) provided with the instrument into a USB C or USB A port on the computer of interest using the appropriate cable supplied with the portable SSD. 2. Navigate to the Cell Microsystems folder on the portable SSD and double-click on the OffTheAIR.msi file to start the installer. 3. Accept the license agreement and click the Install button. 4. If the computer prompts you to enable changes by the installer, click Yes. 5. Click the Finish button once installer completes. 6. The installer should have added an Off the AIR shortcut to the computer Desktop.

Additional Information: 1. When launched, the access icons for the AIR and Off the AIR applications may not appear in the Windows taskbar but will instead be located in the “hidden icons.” Those applications can be accessed by clicking on the “caret” icon (˄) at the right end of the Windows taskbar. 2. The Cell Microsystems folder on the portable SSD also contains an installer (DB.Browser.for.SQLite-3.10.1-win64.exe) for a third-party database browser that can be installed on any computer and used to explore any database record created by the AIR System software. 3. The Cell Microsystems folder on the portable SSD also contains a template Excel file (Plate Map Template.xlsx) that allows the AIR System user to load specific CellRaft addresses into the instrument software for isolation (as described in section 6.12).

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6. System Operation

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6.1. Power on the AIR™ Instrument, Computer, Mouse and Keyboard

The AIR™ System has a single power switch located at the front of the system (Figure 4). If the LED indicator light is emitting blue light the system is already on and depressing the power button again will turn the system off.

The AIR™ System computer has a single power switch located on the lower-right edge of the monitor. Depressing the power button will turn the computer on. No password should be required.

The wireless mouse and keyboard contain single power switches which must be turned on before they can connect to the system computer.

6.2. Key Operational Components

6.2.1. Power Switch

The power button is positioned on the front of the instrument (Figure 4). The LED Indicator will emit blue light when the system is powered and will be dark when the system is off.

6.2.2. Instrument Door

A sliding door provides access to the microscope stage of the AIR™ instrument Figure 4: AIR™ Instrument and exterior components. (Figure 4). The door slides left to open. The AIR System software locks the door in the closed position when the system is actively imaging or actuating mechanical components to ensure both light-tightness for optimal imaging conditions as well as user safety. The door should be opened and closed using the handle, and care should be taken to avoid pinching fingers or other appendages.

6.2.3. AIR™ Instrument Fan and Air Intake

The AIR™ System also includes an internally mounted fan to ensure internal components remain at appropriate temperatures for optimal function. Intake holes on the sides of the instrument should be noted (Figure 4), and nothing should occlude or enter these intakes due to the potential for bodily harm and damage to the instrument.

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6.2.4. AIR™ System Microscope Stage

The AIR™ System deck (Figure 5) consists of a microscope stage with two recessed holders which accommodate standard SBS- format plates. The left position is reserved for the CytoSort Array. The right position is reserved for the collection plate (96-well culture plate, 96-well PCR plate, PCR tube strips) for CellRaft isolation. A full list of validated collection plates can be found in Appendix 1 – Labware Compatibility.

The AIR System will align the deck with the instrument door to facilitate loading and unloading of the CytoSort Array and Figure 5: AIR™ System microscope stage loaded with a CytoSort collection plate. The brightfield illuminator Array and 96-well culture plate for isolation. is moved to the far left of the loading area to avoid interference. Care should be taken not to bump or otherwise perturb the position of the white light illuminator and/or collection wand to avoid sub-optimal performance.

When placing both the array/adapter and collection plate on the deck, care should be taken to not spill any media or other liquid reagents onto the underlying components as this can damage the function of those components.

6.2.5. CytoSort Array Adapter Plate

CytoSort Arrays with a 65-mm diameter form factor are held in place on the deck of the instrument by the CytoSort Array Adapter Plate (Figure 6) included in the AIR System Accessory Kit. The array should be placed with the Keying Arrow’s flat side toward the back of the adapter plate. Matching the flat section on the inner diameter of the adapter plate to the flat side on the outer diameter of the CytoSort Array standardizes the placement of the array from run-to-run. A spring-loaded pin at the front of the adapter applies light pressure to the array to stabilize its location within the adapter. The adapter plate should always be loaded into the left well of the instrument deck and always in the direction of the arrow on the adapter plate.

When prompted by the AIR System software to load the CytoSort Array onto the instrument, Figure 6: AIR™ System CytoSort Array Adapter Plate.

Page 27 of 102 CellRaft AIR™ System User Manual the CytoSort Array can be loaded into the adapter plate then the entire assembly onto the instrument deck; alternatively, the adapter plate can be placed on the deck first with the array placed in the adapter after.

Care should be taken to avoid spilling media or reservoir contents when positioning the adapter plate holding a CytoSort Array on the deck of the AIR™ System. Leaving the array lid on a CytoSort Array during the loading process is likely to mitigate spills.

6.2.6. 96-well culture plate compatibility

The Standard Wand (Figure 7) that is included in the AIR System Accessory Kit is designed to be compatible with most flat-bottom 96-well culture plates which adhere to Society for Laboratory Automation Standards (SLAS) dimensions. For a list of these dimensions, please consult the height and well location standards published by SLAS. Links to these documents are provided here: Height: https://www.slas.org/SLAS/assets/File/ANSI_SLAS_2-2004_HeightDimensions.pdf Well Positions: https://www.slas.org/SLAS/assets/File/ANSI_SLAS_4-2004_WellPositions.pdf A full list of validated collection plates can be found in Appendix 1 – Labware Compatibility.

The Standard Wand is compatible with a standard ethanol or isopropanol wipe of the wand surface for sterilization prior to each use. However, do NOT soak the plastic components of the wand in alcohol for extended periods as it can degrade Figure 7: Schematic of the surfaces. the AIR™ System Standard Wand. It is recommended to fill each well within the collection plate with 100 – 150 L of fluid for efficient CellRaft isolation. Isolation into fluid volumes as low as 50 L is possible, but isolation efficiency may be compromised, resulting in higher than expected numbers of empty wells or wells with two or more CellRafts. Higher volumes can cause the wells to overflow during magnetic wand deposit of the CellRaft. Successful CellRaft isolation (>90% efficiency) has been validated into cell culture media, lysis buffer, and PBS.

The 96-well collection plate should always be loaded into the right position of the instrument deck with the A1 well toward the user and to the left.

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6.2.7. PCR Tube Compatibility

The PCR Tube Wand (Figure 8) that is included in the AIR System Accessory Kit is designed to be compatible with most commercially available 0.2mL PCR tube strips and unskirted or semi-skirted PCR plates. During CellRaft isolation, the PCR tube strips or plates must be loaded into the PCR Tube Adapter Plate (Figure 9) provided in the Accessory Kit. A full list of validated PCR tube strips and 96-well PCR plates can be found in Appendix 1 – Labware Compatibility.

ERROR WARNING: While the PCR Tube Adapter Plate resembles commercially available PCR tube holders, those holders should NOT be used on the AIR System. The AIR System isolation process is precisely calibrated to the height of the PCR

Tube Adapter Plate. Using a commercially available tube holder may damage the magnetic wand or impact the efficiency of CellRaft isolation. Figure 8: Schematic of the AIR™ System PCR Tube Wand. ERROR WARNING: PCR tubes with attached caps and fully skirted PCR plates should NOT be used on the AIR system given that they extend the overall height of the tube / plate and may damage the magnetic wand or impact the efficiency of CellRaft isolation.

The PCR Tube Wand is compatible with a standard ethanol or isopropanol wipe of the wand surface for sterilization prior to each use. However, do NOT soak the plastic components of the wand in alcohol for extended periods as it can degrade the surfaces.

Figure 9: AIR™ System PCR Tube Adapter Plate. It is recommended to fill each PCR tube within the Adapter Plate with at least 2.5 L of fluid for CellRaft isolation. Isolation into fluid volumes as low as 1 L is possible, but collection efficiency will be reduced resulting in higher than expected numbers of empty tubes or tubes with two or more CellRafts. Higher volumes (> 100 L) can cause the tubes to overflow during magnetic wand deposit of the CellRaft. Successful CellRaft isolation (>90% efficiency) has been validated into cell culture media, lysis buffer, and PBS.

It is important to avoid bubbles within the collection fluid as this may affect deposit efficiency. It is recommended with low collection volumes (< 5 L) to centrifuge the PCR tubes or plates prior to loading in the Adapter Plate.

The PCR Tube Adapter Plate should always be loaded into the right position of the instrument deck and always in the direction of the arrows on the adapter plate.

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Collection efficiency in PCR tubes should be experimentally determined by the user as results vary depending on conditions including fluid volume. The collection of CellRafts in the collection vessel can be confirmed using low magnification imaging; see section 6.11.3 of this manual. Contact Cell Microsystems technical support for further information.

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6.3. Experimental Design with the AIR System

The CellRaft AIR System and software provide a suite of capabilities to image, analyze, and select CellRafts for isolation from a variety of CytoSort Array formats into multiple types of collection plates. There are several steps to setting up an experiment on the AIR™ System. Table 3 outlines a few of the more common experimental designs. Cell Microsystems customer support can provide guidance on specific workflow features and how they can be utilized in a given experiment.

To guide the user through these experimental designs, a schematic of the AIR System Software architecture and user interface workflow is shown in Figure 10. The AIR and Off the AIR executables work in concert with one another and are configured around a central Home screen that displays the data components acquired from a given CytoSort Array.

Table 3: Table describing common experimental designs with the CellRaft AIR System.

Experimental Goal Enabling Equipment or Software Feature Collect cells in minimal liquid CellRaft isolation into PCR tubes using PCR Tube Adapter Plate and PCR Tube Wand: volume (e.g. genomic analysis) The AIR System can perform CellRaft isolation into standard 0.2mL PCR tubes or plates and collection volumes of 2.5 – 100 L. For collection volumes below 10L, centrifugation of the PCR tubes before and after isolation is recommended. See section 6.2.7 for further details. Collect cells or colonies for CellRaft isolation into 96-well culture plate using Standard Wand: propagation culture (e.g. The AIR System can perform CellRaft isolation into standard SLAS-format flat-bottom 96-well culture transfection) plates and collection volumes of 100 – 150 L. After isolation, the colony will grow on the CellRaft and over time, onto the floor of the well. Minimize the amount of time the Full Array Scan followed by User Review or Cytometric Image Analysis mode: cells on the CytoSort Array are A Full Array Scan acquires user-configured three-channel fluorescence and brightfield images from outside the incubator every CellRaft within the CytoSort Array. Array loading, imaging setup, and scan execution can generally be performed in less than 15 minutes. After the scan, the CytoSort Array can be returned to the incubator to allow the cells to recover while the user analyzes the images to select CellRafts for downstream isolation. Sort CellRafts containing single cells Full Array Scan followed by Cytometric Image Analysis mode: or colonies based on fluorescence A Full Array Scan acquires user-configured three-channel fluorescence and brightfield images from or brightfield image information every CellRaft within the CytoSort Array. Cytometric Image Analysis mode can be configured to detect single cells or colonies based on fluorescence thresholds or brightfield contrast, then sort and select the detected CellRafts based on fluorescence intensities, brightfield information, and imaging changes over time. Select CellRafts for isolation based Full Array Scan followed by User Review and Selection mode: on user review of acquired images A Full Array Scan acquires user-configured three-channel fluorescence and brightfield images from every CellRaft within the CytoSort Array. User Review and Selection mode can be used to examine the stored images across one or more array scans through virtual navigation around the CytoSort Array and to select single cells or colonies for isolation. Perform a time-course study to Multiple Full Array Scans over time: view the cell dynamics within The user can scan the array multiple times over a desired time course to collect CellRaft images across CellRafts the full array. Using the accumulated images, the software can inform the user about: which colonies started as single cells, changes in fluorescence or brightfield over time, and confluency estimates. If a user chooses to isolate a subset of the CellRafts at each timepoint, the rest of the time course can proceed as desired. Survey the CytoSort Array using User Navigation and Selection mode: real-time imaging and select The user can position the microscope field of view within the CytoSort Array and configure the CellRafts for isolation microscope focus and acquisition parameters for real-time three-channel fluorescence and brightfield images. CellRafts can be selected for isolation directly from the images.

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CellRaft AIR™ System User Manual

software modules software and different of methods CellRaft imaging

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6.4. Hardware and Software Initialization

6.4.1. Software Start-up and System Initialization

To launch the AIR™ System control software, double-click on the AIR shortcut on the Desktop of the AIR System control computer.

Upon software launch, the initialization screen will appear while the software initializes the system hardware (Figure 11).

ERROR WARNING: If the AIR™ System instrument is not powered on or not connected to the computer, the software will prompt that it is unable to locate one or more of the hardware components. Figure 11: AIR System software initialization screen ERROR WARNING: AIR System hardware initialization requires movement of various motorized components within the instrument. If the door of the AIR™ System is left open during initialization, the software will prompt the user to close the door before continuing.

6.4.2. Creating a new database record for a CytoSort Array

After hardware initialization within the AIR software or directly after launch of the Off the AIR software, the software will display the Database Loading screen (Figure 12).

Figure 12. Database Loading screen. NOTE: It is optional to connect a portable solid-state drive (USB 3.1 version 2 or faster) to the computer to create the database record on the SSD rather than the computer hard drive.

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A unique CytoSort Array ID should be entered into the edit box next to the “Search” button. The ID is limited to twelve alphanumeric characters (0-9, A-Z) with no special characters allowed.

After a CytoSort Array ID is entered, the “Search” button is enabled. Once selected, the software searches the computer hard drive and all external drives for a database record to match the Array ID.

When using an Array ID for the first time, a message reading “No databases found, create a new database” will appear.

Selecting the “Create” button move to a series of drop-down menus for the user to select options for the new data record, including the type of array, the drive location for the data record (C:\ for the AIR System computer, other drive letters for external hard drives), and the default type of collection plate.

NOTE: An external SSD is recommended to enable easy transport of data between the AIR System control computer and other computers.

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NOTE: The default plate type selection does not require CellRaft isolation into the selected plate type. The collection plate type to be used for CellRaft isolation can be changed prior to physically isolating CellRafts from the CytoSort Array.

Once all selections are made and the “Continue” button is clicked, the software creates a new database record associated with the Array ID in the selected storage location. The software “Home” screen will then appear (Figures 13 and 14 in section 6.5).

6.4.3. Loading an existing database record for a CytoSort Array

NOTE: It is optional to connect a portable solid-state drive (USB 3.1 version 2 or faster) to the computer to access a database record on the SSD rather than the computer hard drive.

An existing database can be loaded from the Database Loading screen (Figure 12), in one of two ways: 1. The unique ID associated with the CytoSort Array and its data record can be entered into the edit box next to the “Search” button. Once the ID is entered, the “Search” button is enabled. Once selected, the software searches the computer hard drive and all external drives for a database record to match the Array ID. The matching database records will be displayed in a list. The desired database record should be selected and the “Open” button clicked.

2. The “View List” button can be selected and the database records from the AIR System computer hard drive and all external hard drives will be listed in a table for selection. The desired database record should be selected and the “Open” button clicked.

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Once the existing database record is loaded, the software “Home” screen (Figures 13 and 14 in section 6.5) will appear with all full array scan data, mapped collection plates, cytometric analyses, and controls to export experimental reports.

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6.5. AIR System Software Home Screens

After a database record is loaded or a new one is created, the AIR and Off the AIR software modules display their respective Home screens (Figures 13 and 14). The Home screens for each module are designed to provide user access to AIR System hardware and data analysis functionality that is available based on the software module and the contents of the data record.

As depicted in Figure 13, the AIR software Home screen allows the user to launch system functions that require the AIR instrument, such as: • selecting CellRafts for isolation in User Navigation and Selection mode • executing a Full Array Scan of a CytoSort Array, and • isolating selected CellRafts into a collection plate.

As depicted in Figure 14, the Off the AIR software Home screen allows the user to perform data analysis functions on Full Array Scan data, such as: • selecting CellRafts for isolation in Cytometric Image Analysis mode and • selecting CellRafts for isolation in User Review and Selection mode and use the CellRaft Organizer screen to track, filter, and review images from CellRafts throughout an experiment.

The user is able to switch between the Home screens of the two software applications using the “Off the AIR” and “Return to AIR” buttons on their respective Home screens. However, the “Return to AIR” button is only visible in the Off the AIR application if the Off the AIR Home screen was accessed using the “Off the AIR” button from the AIR software Home screen, since that guarantees that the Off the AIR module was accessed from a computer that is connected to the instrument.

The AIR and Off the AIR Home screens are laid out identically, with: • CytoSort Array information displayed at the top • A tabbed central console for displaying full array scan data, mapped collection plates, cytometric analyses, and controls to export experimental reports • Button options or plate schematics on the right margin specific to the current module and selected console tab • Global buttons at the bottom to close the current database record and switch between Home screens.

6.5.1. AIR software module Home screen

The controls for the AIR software Home screen (Figure 13) allow the user to launch system functions that require the AIR instrument, such as: • selecting CellRafts for isolation in User Navigation and Selection mode (section 6.6) • executing a Full Array Scan of a CytoSort Array (section 6.7) • isolating selected CellRafts into a collection plate (section 6.11).

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Figure 13. Home screen for the AIR software module.

Functionality for each of the controls on the AIR module Home screen is outlined below:

CytoSort Array information: The top aspect of the Home screen lists descriptive information regarding the CytoSort Array associated with the current database record, provided by the user or inherent to the array. That information includes the unique alphanumeric ID assigned to the CytoSort Array, the array type (e.g. 200m single-reservoir), and any array description provided by the user. The Description field can be modified by selecting the “Edit Mode” checkbox below the Tabbed Console.

Tabbed Console and Tab-Specific Function Buttons: A tabbed console in the central aspect of the Home screen provides access to major components of the database record and button options on the right margin allow the user to launch software functions (described in subsequent sections) that are specific to the currently selected console tab and require the AIR instrument.

Full Array Scans: Provides a list of the Full Array Scans acquired from the CytoSort Array to date. The columns in the table include a description of the scan (Description), when it was executed (Start Time), the location of the imaging data (Image Folder), the imaging channels acquired (Channel), and the acquisition settings during the scan (Binning, Gain, and Exposure). The Description field can be modified by the user by selecting the “Edit Mode” checkbox below the Tabbed Console.

When the Full Array Scans tab is selected, two tab-specific function buttons are visible. The “Full Array Scan” button launches the process to acquire a Full Array Scan from the CytoSort Array (as described in section 6.7). The “Real-Time User Selection” button launches User Navigation and

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Selection mode (as described in section 6.6), which allows the user to select CellRafts for isolation via real-time images from the AIR System.

Isolation Plate Maps: Provides a list of the collection plates mapped by the user to date. The columns in the table include a description of the plate map (Description), the type of collection plate to be used during CellRaft isolation (Collection Plate Type), the date/time range over which the CellRafts were selected for isolation (Selection Timespan), and the date/time range over which the CellRafts were isolated (Isolation Timespan). The table row for the currently selected Isolation Plate Map also contains an “Isolate” button that will initiate the CellRaft Isolation process as described in section 6.11.

There are no tab-specific buttons in the right margin; instead, a schematic of the currently selected Isolation Plate Map is displayed with the color groups. The circles around each collection plate well in the schematic indicate the isolation status of the well: a dashed ring indicates that the CellRaft has not yet been isolated; solid rings indicate that isolation was performed and the color of the ring indicates its result as described in section 6.11. The names of the color groups can be edited by the user by selecting the “Edit Mode” checkbox below the Tabbed Console.

By selecting the “Edit Mode” checkbox below the Tabbed Console, the user can modify: • The Description field for each plate map in the list • The Collection Plate Type for each plate map in the list using the drop-down menu of compatible plate types • The “A1 rinse” option for each plate map in the list – clicking the sprinkler head at the right end of the row toggles between the enabled and disabled states. If water is coming out of the sprinkler head, the A1 rinse option is enabled. If enabled, during the CellRaft isolation process the AIR System will immerse the tip of the magnetic collection wand in the A1 well of the collection plate – which can be filled with buffer, ethanol, or some other rinsing solution – before the first isolation and after every isolation to prevent cross- contamination between collection wells.

NOTE: If a CellRaft is mapped to the A1 position within the collection plate and the A1 rinse option is enabled, the CellRaft mapped to position A1 will not be isolated.

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The “Access Wand” button below the “Edit Mode” checkbox moves the collection wand attachment plate to a middle position within the door aperture for easier access to remove the collection wand after isolation. To detach the wand from the attachment plate, simply squeeze the compression key on the blue connector and pull downward to slide the connector from the attachment plate. Additional details can be found in section 6.11.2.

Global Buttons: The “Close Record” button closes the current database record and returns to the Database Loading screen depicted in Figure 12 (section 6.4). The “Off the AIR” button launches the Off the AIR software module and displays its Home screen as described in section 6.5.2.

6.5.2. Off the AIR software module Home screen

The controls for the Off the AIR software Home screen allow the user to launch data analysis functions that do not require the AIR instrument (Figure 14), such as: • selecting CellRafts for isolation in Cytometric Image Analysis mode (section 6.9) • selecting CellRafts for isolation in User Review and Selection mode (section 6.8). and use the CellRaft Organizer screen (section 6.10) to track, filter, and review images from CellRafts throughout an experiment.

Functionality for each of the controls on the Off the AIR module Home screen is outlined below:

CytoSort Array information: The top aspect of the Home screen lists descriptive information regarding the CytoSort Array associated with the current database record, provided by the user or

Figure 14. Home screen for the Off the AIR software module.

Page 40 of 102 CellRaft AIR™ System User Manual inherent to the array. That information includes the unique alphanumeric ID assigned to the CytoSort Array, the array type (e.g. 200m single-reservoir), and any array description provided by the user. The Description field can be modified by selecting the “Edit Mode” checkbox below the Tabbed Console.

When the Full Array Scans tab is selected, two tab-specific function buttons are visible. The “User Review and Selection” button launches User Review and Selection mode (as described in section 6.8) which allows the user to select CellRafts for isolation from inspection of images acquired from one or more full array scans. The “Cytometric Review” button launches Cytometric Image Analysis mode (as described in section 6.9), which allows the user to select CellRafts for isolation via cytometric image analysis of the imaging data. The “CellRaft Organizer” button launches the CellRaft Organizer screen which displays the CellRafts from the CytoSort Array in table form and allows the user to filter the table based on a variety of database information, review CellRaft images, and create or modify collection plate maps (section 6.10), all designed to allow users to easily track and evaluate CellRafts throughout an experiment.

By selecting the “Edit Mode” checkbox below the Tabbed Console, the user can: • Edit the Description field for each plate map in the list

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• Select the Collection Plate Type for each plate map in the list using the drop-down menu of compatible plate types • Erase all CellRaft assignments from the currently selected collection plate using the purple eraser icon • Delete the currently selected collection plate from the list using the red trashcan icon.

Marker Gating: Lists the marker gates promoted by the user using Cytometric Imaging Analysis mode as described in section 6.9. By selecting the “Edit Mode” checkbox below the Tabbed Console, the user can: • Edit the Description field for each marker gate in the list • Delete the currently selected marker gate from the list using the red trashcan icon.

Report Generation: Lists the full array scans, isolation plate maps, and promoted marker gates to date and allows the user to select them for export to a report spreadsheet compatible with Microsoft Excel® as described in section 6.13. Each item listed can be selected for inclusion in the report or deselected for exclusion. The “Toggle All” checkbox below the Tabbed Console can be used to select or deselect all options. Clicking the “Generate Report” button generates the report and replaces the button with a hyperlink to the directory containing the report and image data. The directory is created within the directory containing the database record on the computer hard drive or external SSD. The reports directory names are timestamped to enable export of multiple report combinations from a single database record and multiple timepoints within the experimental workflow.

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Global Buttons: The “Close Record” button closes the current database record and returns to the Database Loading screen depicted in Figure 12. If the Off the AIR software module was accessed from the AIR software module, the “Return to AIR Module” button returns to the Home screen of the AIR software module as described in section 6.5.1.

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6.6. Selecting CellRafts for Isolation using User Navigation and Selection Mode

During User Navigation and Selection mode, the system effectively operates as an automated microscope, allowing the user to examine real-time fluorescence and brightfield images of the CytoSort Array, identify which CellRafts contain a single cell or colony of interest, and select which should be isolated for downstream analysis.

The system allows software-controlled navigation within the CytoSort Array while brightfield and fluorescence imaging parameters can be adjusted in real time. CellRafts can be selected for isolation directly from the real-time images, can be labeled as part of a cell or phenotype population, and can be assigned to a specific well or tube in the collection vessel.

In general, the steps to using User Navigation and Selection are accomplished using a single Imaging screen of the user interface software. Once one or more plates of CellRafts are selected for isolation, the user can return to the Home screen where the mapped collection plate(s) can be selected for isolation (see section 6.11).

NOTE: This mode is only compatible with the AIR software, not with Off the AIR.

6.6.1. Entering User Navigation and Selection mode

From the AIR software Home screen and with the “Full Array Scans” tab selected, clicking the “User Navigation” button will launch User Review and Selection mode.

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The software will prompt the user to place the CytoSort Array associated with the database record into the CytoSort Array Adapter Plate (NOTE: the HexaQuad array format does not require the adapter plate) and load it into the left well of the AIR System stage. Guide railings in the stage well assure proper positioning of the CytoSort Array. The Adapter Plate or HexaQuad Array should be inserted so the arrow(s) on the plate points toward the interior of the system.

Upon hitting the “Continue” button, the AIR System will calibrate the position of the CytoSort Array for navigation during imaging.

6.6.2. Real-Time Imaging in User Navigation and Selection mode

Once array calibration is complete, the “Imaging” screen will appear (Figure 15). The controls on the Imaging screen are designed to allow the user to image the CellRafts within the CytoSort Array in real time as if using an automated microscope. The AIR system constantly images a region-of-interest on the CytoSort Array; the region of interest can be moved, the microscope focus can be adjusted, and the image channel acquisition parameters can be modified. Images from individual imaging channels or individual CellRafts can be displayed in full-screen mode, and CellRafts can be selected for isolation directly from any displayed image.

Functionality of each of the controls in the Imaging screen is outlined below:

Real-Time Channel Images: Real-time images from the user-selected field of view on the CytoSort Array in each of the three fluorescence and brightfield channels, using the settings defined in the

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Figure 15: Imaging screen as part of User Navigation and Selection mode.

Imaging Parameter Control Panel. Clicking on the name of a given imaging channel will highlight it and allow the user to change its name.

Image Intensity Histograms: Real-time histograms of image intensities across all pixels within the real-time fluorescence and brightfield images. The controls on the histograms allow the user to adjust the contrast within the images for better visualization; however, adjustment does not impact the underlying raw imaging data.

Focus Control: The AIR System performs an autofocus every time the microscope field of view is changed. Clicking the left and right arrows within the focus control adjusts the microscope focus from the autofocus plane. The outsides of the arrows can be used for coarse adjustment (40m) and the insides for fine adjustment (2.5m). Clicking the “Auto” button returns the microscope focus to the autofocus plane. Selecting the “lock” checkbox moves the focus to the user-selected offset from the autofocus plane after navigating to a new field of view.

Array Navigation: Allows the user to adjust the region of interest shown in the real-time images and histograms. The arrows move the region of interest left, right, up, down or diagonally by one field of view (approximately 1.2 mm X 1.5 mm). A single click within the Array Navigation Box will also adjust the region of interest to that field of view. The navigation tool varies based on array format:

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Single Reservoir Quad Reservoir HexaQuad

Imaging Parameter Control Panel:

Channel Selection: Clicking the name of a given imaging channel toggles between the “On” and “Off” states for that imaging channel for real-time display. When in full-screen mode, clicking on the name allows the user to switch quickly between imaging channels.

Binning: Clicking the “1x1” button for a given imaging channel toggles between 1x1, 2x2, and 4x4 selections for binning the camera pixels during acquisition. Binning 1x1 reads out all camera pixels independently, while 2x2 binning sums the pixel intensity across each set of four pixels within the camera and reads it out as a single pixel. The increased signal intensity comes at the cost of reduced spatial resolution.

Gain: Clicking the left and right arrows for a given imaging channel adjusts the multiplicative gain applied to the signal intensities within the image between 1 and 10. It should be noted that increasing the gain multiplies all pixel intensities, including background fluorescence and noise.

Exposure: Clicking the left and right arrows for a given imaging channel adjusts the amount of time the camera is exposed to the fluorescence or trans-illuminated light during image capture. Longer exposure times result in more signal captured from the cells and therefore a “brighter” image. Again, changes in exposure equally affect capture of background fluorescence. The exposure can be set between 1 millisecond and 2 seconds, with the outsides of the arrows used for coarse adjustment and the insides for fine adjustment.

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Image Zoom Options: The software provides multiple options to better visualize the real-time images of the entire field of view or for a single CellRaft.

Zoom in on one field of view in a single imaging channel (Figure 16):

Clicking on the Zoom icon on any of the imaging channels shows a full-screen display of that imaging channel. Quickly toggle between the imaging channels using the Channel Selection buttons in the Imaging Parameter Control Box.

Figure 16: Full screen view of the GREEN fluorescence imaging channel within User Navigation and Selection mode.

Show the image of an individual CellRaft in place of the Array Navigation Box (Figure 17): Clicking on a CellRaft of interest in any imaging channel displays an enlarged version of the CellRaft image in place of the Array Navigation tools. Dismiss the individual CellRaft image with the red X to re-display the Array Navigation tools.

Figure 17: Enlarged image of selected CellRaft within User Navigation and Selection mode.

Show a full-screen image of an individual CellRaft (Figure 18): When displaying a single imaging channel in full-screen mode and an individual CellRaft is selected for display in place of the Array Navigation tools, clicking on the double-arrow icon swaps the single CellRaft and full field of view image positions to display the CellRaft in full-screen mode. Clicking the icon again swaps them back to their original Figure 18: Full screen view of an individual CellRaft in User Navigation and Selection mode. positions. While the field-of-view image is displayed in place of the Array Navigation Box, CellRafts can still be selected from the image for individual display, and the Channel Selection buttons in the Imaging Parameter Control Box can still be used to toggle between the imaging channels.

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6.6.3. CellRaft Selection for Isolation in User Navigation and Selection mode

Clicking on a CellRaft of interest in any displayed image within the Imaging screen selects and outlines the CellRaft with a box (see Figures 17 and 18). The user can then click on any unoccupied well location within the collection plate map to assign the selected CellRaft to that collection location during the release and transfer process (see section 6.11).

At the time of assignment into the collection plate: • Real-time images of the selected CellRaft are acquired and saved from all active imaging channels using the current focus and acquisition settings for inclusion in the database record and any experimental reports (see section 6.13). • The well in the collection plate schematic is filled with the CytoSort Array address of the CellRaft assigned to it. • The selected CellRaft is assigned to the Active Color Group, controlled by the colored circles in the lower-right of the screen. Clicking on the colored circle for a given color group selects that group as the Active Color Group; a bright green circle is used to indicate the selection. Clicking on the group name allows the user to assign unique names to each color group.

NOTE: During the CellRaft isolation process, the AIR System can be configured to immerse the magnetic collection wand into the A1 well between CellRaft isolations to prevent cross-contamination between wells in the collection plate. If this option is desired, the user should leave the A1 well of the collection plate schematic unassigned – a diagonal blue line serves as a reminder. If the A1 well is assigned and the rinse option is enabled, the CellRaft assigned to the A1 well will not be isolated.

Reviewing the Collection Plate: Clicking on an occupied well in the collection plate schematic overlays an Image Review window to allow the user to inspect the images saved for the CellRaft assigned to that well (Figure 19). A second left-click in the collection well dismisses the Image Review window.

Within the Image Review window, the user can: • Use the arrow buttons to cycle the Figure 19: Image Review window in User Navigation and positions of the various imaging Selection mode. channels. The largest image position in the window contains a pixel histogram with controls to adjust the contrast within the image for better visualization; however, contrast adjustment does not impact the underlying raw images which are saved as part of the data report. • Use the thumbs up / down and star-rating controls to assign ratings to the selected CellRaft to more easily track it over the course of an experiment. CellRafts can be filtered based on either rating control using the CellRaft Organizer as described in section 6.10.

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Figure 20: Collection Plate Menu options in User Navigation CellRafts may instead be deleted from the and Selection mode. collection plate by left-clicking on their position in the plate, right-clicking on the same location to invoke the “trashcan” icon, then confirming deletion by right-clicking a second time.

Mapping multiple collection plates: Additional collection plates can be added for mapping by clicking on the Collection Plate Menu icon and selecting the green plus sign from the options (Figure 20). The user can toggle between plates using the left and right arrow keys.

Erasing or deleting collection plates: All CellRaft assignments from the currently viewed collection plate can be erased using the purple eraser icon from the Collection Plate Menu. Similarly, the currently viewed collection plate can be deleted using the red trashcan icon.

Return to Home tab: Clicking the “Return to Home” tab at the top of the screen returns to the AIR software module Home screen with any mapped collection plates now included in the database record and listed under the “Isolation Plate Maps” tab, where they can be selected for isolation (see section 6.11).

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6.7. Acquiring a Full Array Scan on a CytoSort Array

During a full array scan, the CytoSort Array is scanned in its entirety in any combination of the four imaging channels including brightfield and the three fluorescence channels.

In general, the steps of a full array scan comprise: 1) setting the imaging parameters; 2) starting the scan; and 3) executing the scan.

NOTE: This mode is only compatible with the AIR software, not with Off the AIR.

6.7.1. Entering Full Array Scan mode

From the AIR software Home screen and with the “Full Array Scans” tab selected, clicking the “Full Array Scan” button will initiate the process to image every CellRaft within the CytoSort Array.

The software will prompt the user to place the CytoSort Array associated with the database record into the CytoSort Array Adapter Plate and load them into the left position of the AIR System stage. The CytoSort Array can either be placed in the adapter plate before or after inserting the adapter plate into its deck position (see section 6.2.5). Guide railings in the stage well assure proper positioning of the CytoSort Array. The Adapter Plate should be inserted so the arrow on the plate points toward the interior of the system.

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Upon hitting the “Continue” button, the AIR System will calibrate the position of the CytoSort Array for navigation during imaging.

6.7.2. Imaging Setup for a Full Array Scan

Once array calibration is complete, the “Imaging Setup” screen will appear (Figure 21), which allows the user to set the imaging parameters independently for each of the four imaging channels during an automated scan of every CellRaft within the CytoSort Array. The images acquired during the scan are stored in the database record and are ultimately used to identify CellRafts of interest for isolation.

Functionality of each of the controls in the Imaging Setup screen is outlined below:

Real-Time Fluorescence Images: Real-time images from the user-selected field of view on the CytoSort Array in each of the three fluorescence channels, using the settings defined in the Imaging Parameter Control Panel. For a multi-reservoir array (e.g. Quad or HexaQuad), the images are only displayed when the location of the current field of view coincides with the Reservoir Selection as described below.

Fluorescence Intensity Histograms: Real-time histograms of fluorescence intensities across all pixels within the real-time fluorescence images. The controls on the histograms allow the user to adjust the contrast within the images for better visualization; however, adjustment does not impact the underlying raw image files. For a multi-reservoir array (e.g. Quad or HexaQuad), the histograms are only displayed when the location of the current field of view coincides with the Reservoir Selection as described below.

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Figure 21: Imaging Setup screen as part of Full Array Scan mode.

Array Navigation: Allows the user to adjust the region of interest shown in the real-time fluorescence images and histograms. The arrows move the region of interest left, right, up, down or diagonally by one field of view (approximately 1.2 mm X 1.5 mm). A single click within the Array Navigation Box will also adjust the region of interest to that field of view. The navigation tool varies based on array format:

Single Reservoir Quad Reservoir HexaQuad

Imaging Parameter Control Panel:

Reservoir Selection: For a multi-reservoir array (e.g. Quad or HexaQuad), clicking on a single reservoir, clicking on multiple reservoirs while holding the CTRL key, or clicking on the “ALL” button allows the user to set the imaging parameters in the control panel individually for each

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reservoir, for a subset of reservoirs, or for all reservoirs simultaneously. The shading of selected reservoirs within the tool is light gray when the reservoir corresponds to the location of the real- time field of view and red when it does not. The font color of the reservoir label corresponds to the Cell Counting Channel (described below) assigned to that reservoir.

Channel Name: Clicking on the name of a given fluorescence channel will highlight it and allow the user to change its name.

Channel Selection: Clicking the “On” button for a given fluorescence channel toggles between the “On” and “Off” states for that imaging channel for inclusion or exclusion during the full array scan.

Binning: Clicking the “1x1” button for a given fluorescence channel toggles between 1x1, 2x2, and 4x4 selections for binning the camera pixels during acquisition. Binning 1x1 reads out all camera pixels independently, while 2x2 binning sums the pixel intensity across each set of four pixels within the camera and reads it out as a single pixel. The increased signal intensity comes at the cost of reduced spatial resolution.

Gain: Clicking the left and right arrows for a given fluorescence channel adjusts the multiplicative gain applied to the signal intensities within the image between 1 and 10. It should be noted that increasing the gain multiplies all pixel intensities, including background fluorescence and noise.

Exposure: Clicking the left and right arrows for a given fluorescence channel adjusts the amount of time the camera is exposed to the fluorescence light during image capture. Longer exposure times result in more signal captured from the cells and therefore a “brighter” image. Again, changes in exposure equally affect capture of background fluorescence. The exposure can be set between 1 millisecond and 2 seconds, with the outsides of the arrows used for coarse adjustment and the insides for fine adjustment.

Cell Counting Channel: Specifies which of the fluorescent imaging channels is used during Cytometric Analysis mode to identify single cells within the CellRafts. The selection is also indicated by the font color of the reservoir label within the Reservoir Selection tool (described above). For most accurate results, the cell counting channel should correlate to a nuclear stain of all cells on the CytoSort Array. See section 4.3.3 for suggestions of vital dyes which provide reliably strong nuclear signal that have been validated for use on the AIR System by Cell Microsystems.

Array Scanning tab: Once the parameters are set, clicking the “Array Scanning” tab at the top of the screen will move to the Array Scanning screen, where the user can initiate the full array scan.

Return to Home tab: Clicking the “Return to Home” tab at the top of the screen returns to the Home screen (see section 6.5) without executing the full array scan.

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6.7.3. Executing the Full Array Scan in the Array Scanning screen

Within the Array Scanning screen (Figure 22), clicking the “Start Scan” button initiates image acquisition from every CellRaft within the CytoSort Array using the imaging parameters set in the Imaging Setup screen. On this screen, the user also has the option to enable Adaptive Focus during the scan, in which images are acquired at multiple focus locations for each field of view. The focus image with maximum contrast is determined individually for each CellRaft within the field of view and those images are stitched together to create a mosaic image of maximum focus. Invoking Adaptive Focus increases the scan duration roughly 2-3 fold depending on the number of channels enabled and their exposure times.

During the scan, the current field of view and its four-channel images are displayed as acquired. Clicking the “Pause Scan” button will allow the user to toggle between pausing and resuming the scan. The user also has the option to return to the Imaging Setup screen while the scan is paused to adjust the imaging parameters for one or more imaging channels or reservoirs; however, doing so erases the imaging data acquired during the current full array scan.

Once the scan is complete, the software will return to the “Home” page with the array scan now included in the database record and listed under the “Full Array Scans” tab. The scan data can now be used for analysis using Cytometric Analysis or User Review and Selection modes (described in subsequent sections).

NOTE: The imaging data from a Full Array Scan is saved in a sub-directory created by the AIR System software within the directory containing the database record on the computer hard drive (C:\CMSData\DatabaseName\) or external SSD (X:\CMSData\DatabaseName\). The imaging data is saved as full field of view images in Tagged Image Format (TIF) which can be imported into ImageJ or any other image analysis software for display or further analysis. The image file names follow the naming convention, RRCCI.tiff, where RR corresponds to the row index within the CytoSort Array of the top-left CellRaft in the field of view, CC corresponds to the column index of the top-left CellRaft in the field of view, and I corresponds to the imaging channel – A for a brightfield image, B/G/R for a blue/green/red fluorescence image, respectively. Refer to the CytoSort Array User Manual for more information regarding the row and column addresses of the CellRafts.

Figure 22: Array Scanning screen as part of Full Array Scan mode.

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6.8. User Review and Selection Mode of Full Array Scan Data

After one or more Full Array Scans are acquired from a CytoSort Array, there are two methods to analyze the imaging data to select CellRafts for isolation: User Review and Selection mode (section 6.8) and Cytometric Image Analysis mode (section 6.9).

In User Review and Selection mode, images collected during a Full Array Scan are displayed as the user virtually navigates around the CytoSort Array, allowing the user to identify CellRafts containing single cells or colonies of interest, selecting CellRafts for isolation, and assigning them positions within the collection vessel. The user interface and controls are similar to those described within User Navigation and Selection Mode, but instead of actively collecting real-time images, the software displays the images collected during one or more Full Array Scans.

6.8.1. Entering User Review and Selection mode

From the AIR software module Home screen, clicking the “Data Analysis Module” button will transition to the Off the AIR software Home screen.

The “Full Array Scans” tab lists each scan that has been acquired from the array.

NOTE: Green font indicates that the imaging data from a given array scan was successfully loaded from the database record. Red font indicates that the imaging data was not found – contact Cell Microsystems customer support if needed.

Once all full array scans of interest for review are selected using the checkboxes to the left, clicking the “User Review and Selection” button will launch the Imaging screen.

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NOTE: Since User Review and Selection mode is accessed through the Off the AIR software module, the analysis can be performed from any computer with the Off the AIR software installed and with access to the database record.

6.8.2. Image Review in User Review and Selection mode

The controls on the Imaging screen (Figure 23) are designed to allow the user to easily review the CellRaft images acquired during the selected full array scans. The controls are similar to the Imaging screen from User Navigation and Selection mode (section 6.6), allowing the user to move the displayed region of interest throughout the CytoSort Array, to display the images from individual imaging channels or individual CellRafts in full-screen mode, and to select CellRafts for isolation directly from any displayed image. The differences in controls between the two modes include: 1. Microscope focus control: Since real-time images are not acquired during Image Review and Selection mode, the microscope focus cannot be adjusted. 2. Imaging parameters: Since real-time images are not acquired during Image Review and Selection mode, the imaging parameters cannot be adjusted.

Figure 23: Imaging screen as part of User Review and Selection mode.

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3. Timepoint control: Since multiple full array scans can be selected for review during Image Review and Selection mode, unique controls allow the user to select an individual timepoint for display or to loop across all scans.

Functionality of each of the controls in the Imaging screen is outlined below:

Full Array Scan Channel Images: Images from the timepoint of interest and user-selected field of view on the CytoSort Array in each of the three fluorescence and brightfield channels. Clicking on the name of a given imaging channel will highlight it and allow the user to change its name.

Image Intensity Histograms: Histograms of image intensities across all pixels within the displayed fluorescence and brightfield images. The controls on the histograms allow the user to adjust the contrast within the images for better visualization; however, contrast adjustment does not impact the underlying raw images which are saved as part of the data report.

Timepoint Control: The slider on the timepoint control allows the user to adjust which full array scan is selected for display in the Channel Images. Clicking the “play” button loops the Channel Images through all full array scans selected for review from the Home screen. While looping, the speed of the loop can be adjusted using the “speedometer” control by dragging the “needle” within the control.

Array Navigation: Allows the user to adjust the region of interest shown in the displayed images and histograms. The arrows move the region of interest left, right, up, down or diagonally by one field of view (approximately 1.2 mm X 1.5 mm). A single click within the Array Navigation Box will also adjust the region of interest to that field of view. The navigation tool varies based on array format:

Single Reservoir Quad Reservoir HexaQuad

NOTE: Array navigation is significantly faster in User Review and Selection mode compared to User Navigation and Selection mode since the software is not forced to wait for movement of the microscope stage to access and display the images.

Channel Selection: Clicking the name of a given imaging channel toggles between the “On” and “Off” states for that imaging channel for display. When in full-screen mode, clicking on the name allows the user to switch quickly between imaging channels.

Image Zoom Options: The software provides multiple options to better visualize the images of the entire field of view or for a single CellRaft.

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Zoom in on one field of view in a single imaging channel (Figure 24): Clicking on the Zoom icon on any of the imaging channels shows a full-screen display of that imaging channel. Quickly toggle between the imaging channels using the Channel Selection buttons.

Figure 24: Full screen view of the GREEN fluorescence imaging channel within User Navigation and Selection mode.

Show the image of an individual CellRaft in place of the Array Navigation Box (Figure 25): Clicking on a CellRaft of interest in any imaging channel displays an enlarged version of the CellRaft image in place of the Array Navigation tools. Dismiss the individual CellRaft image with the red X to re-display the Array Navigation tools.

Figure 25: Enlarged image of selected CellRaft within User Navigation and Selection mode.

Show a full-screen image of an individual CellRaft (Figure 26): When displaying a single imaging channel in full-screen mode (Figure 24) and an individual CellRaft is selected for display in place of the Array Navigation tools (Figure 25), clicking on the double- arrow icon swaps the single CellRaft and full field of view image positions to display the CellRaft in full-screen mode (Figure 26). Clicking the double-arrow icon again swaps them back to their original positions. While the field-of- Figure 26: Full screen view of an individual CellRaft in User view image is displayed in place of the Navigation and Selection mode. Array Navigation Box, CellRafts can still be selected from the image for individual display, and the Channel Selection buttons can still be used to toggle between the imaging channels.

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6.8.3. CellRaft Selection for Isolation in User Review and Selection mode

Clicking on a CellRaft of interest in any displayed image within the Imaging screen selects and outlines the CellRaft with a box (see Figures 25 and 26). The user can then click on any unoccupied well location within the collection plate map to assign the selected CellRaft to that collection location during the release and transfer process (see section 6.11).

At the time of assignment into the collection plate: • Images of the selected CellRaft are saved from all imaging channels for inclusion in the database record and any experimental reports (see section 6.13). • The well in the collection plate schematic is filled with the CytoSort Array address of the CellRaft assigned to it. • The selected CellRaft is assigned to the Active Color Group, controlled by the colored circles in the lower-right of the screen. Clicking on the colored circle for a given color group selects that group as the Active Color Group; a bright green circle is used to indicate the selection. Clicking on the group name allows the user to assign unique names to each color group.

Reviewing the Collection Plate: Clicking on an occupied well in the collection plate schematic overlays an Image Review window to allow the user to inspect the images saved for the CellRaft assigned to that well (Figure 27). A second left-click in the collection well dismisses the Image Review window.

Within the Image Review window, the user can:

• Use the arrow buttons to cycle the positions of the various imaging Figure 27: Image Review window in User Navigation and Selection mode. channels. The largest image position in the window contains a pixel histogram with controls to adjust the contrast within the image for better visualization; however, contrast adjustment does not impact the underlying raw images which are saved as part of the data report. • Use the thumbs up / down and star-rating controls to assign ratings to the selected CellRaft to more easily track the CellRaft over the course of an experiment. CellRafts can be filtered based on either rating control using the CellRaft Organizer as described in section 6.10.

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CellRafts may instead be deleted from the collection plate by left-clicking on their position in the plate, right-clicking on the same location to invoke the “trashcan” icon, then confirming deletion by right- clicking a second time.

Mapping multiple collection plates: Additional collection plates can be added for mapping by clicking on the Collection Plate Menu icon and selecting the green plus sign from the options (Figure 28). The user can toggle between plates using the left and right arrow keys.

Erasing or deleting collection plates: All CellRaft assignments from the currently viewed collection plate can be erased using the purple Figure 28: Collection Plate Menu options in User Review and eraser icon from the Collection Plate Menu. Selection mode. Similarly, the currently viewed collection plate can be deleted using the red trashcan icon.

Return to Home tab: Clicking the “Return to Home” tab at the top of the screen returns to the Off the AIR module Home screen with any mapped collection plates now included in the database record and listed under the “Isolation Plate Maps” tab, where they can be selected for isolation (see section 6.11).

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6.9. Cytometric Image Analysis Mode of Full Array Scan Data

The steps for selecting CellRafts for isolation in Cytometric Mode include: 1) CellRaft Gating to perform a preliminary filter of CellRafts based on number of cells as detected in brightfield or using a nuclear fluorescent stain or based on fluorescence intensity thresholds 2) Marker Gating to further divide the gated CellRaft population into sub-populations of interest 3) Plate Mapping to select individual CellRafts from the gate populations and map them to positions within the collection vessel for deposition.

During CellRaft Gating, the Full Array Scan imaging data are used to apply a preliminary filter to the CellRaft population in the CytoSort Array based on the number of cells present (as detected using fluorescence or brightfield) and/or the CellRaft fluorescence intensities. During Marker Gating, additional brightfield and fluorescence metrics calculated for each CellRaft are charted on a scatterplot. Cytometric gates can be applied to the scatterplot to create sub-population(s) of CellRafts that have desirable fluorescence or brightfield properties such as relative fluorescence intensity, number of cells as detected in brightfield, percent confluency on the CellRaft, and changes in these metrics between timepoints (i.e. full array scans). Finally, during Plate Mapping, individual CellRafts can be randomly or explicitly selected from the marker gate populations for isolation and assigned to locations within the collection plate.

NOTE: In one CellRaft Gating mode, the AIR system can identify CellRafts containing single cells by detection of a nuclear fluorescence signal applied to all cells in the experiment. The fluorescent nuclei are used by the AIR System software to effectively count cells Imaging Nominal Imaging Dye Concentration Exposure Channel on the CytoSort Array and SYTO® 13 Green 5 μM 2 ms Green automatically identify which SYTO® 17 Red 5 μM 15 ms Red CellRafts contain a single cell. SYTO® 41 Blue 5 M 50 ms Blue Strong nuclear localization Hoechst 33342 1.6 μM (1 μg/mL) 20 ms Blue promotes a more accurate count Table 2: List of example dyes which can be used to stain nuclei in each of the of CellRafts which contain a single standard AIR System fluorescent imaging channels. Note that any of the cell. Any of the three fluorescent fluorescent channels can be used to count the cells during Cytometric Analysis. channels can be used for the Cell Counting Channel. See Table 2 (reproduced here from section 4.3.3) for suggestions of vital dyes which provide reliably strong nuclear signal that have been validated for use on the AIR System by Cell Microsystems. Refer to section 4.3.3 for additional details on selecting appropriate stains and fluorophores to reveal cellular nuclei and enable automated detection of CellRafts with single cells.

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6.9.1. Entering Cytometric Image Analysis mode

From the AIR software module Home screen, clicking the “Off the AIR” button will transition to the Off the AIR software Home screen.

The “Full Array Scans” tab lists each scan that has been acquired from the array. Any or all of these full array scans can be included in the Cytometric Image Analysis.

Once all full array scans of interest for review are selected using the checkboxes to the left, clicking the “Cytometric Image Analysis” button will launch the CellRaft Gating screen.

NOTE: Since Cytometric Image Analysis mode is accessed through the Off the AIR software module, the analysis can be performed from any computer with the Off the AIR software installed and with access to the database record.

NOTE: If the workflow is predicated on gating CellRafts that contain or start from single cells, it is recommended to acquire a full array scan shortly after seeding cells on the CytoSort Array, before they have had the opportunity to proliferate.

6.9.2. CellRaft Gating in Cytometric Image Analysis mode

The first step in the Cytometric Image Analysis process – CellRaft Gating – is designed to identify CellRafts whose contents meet preliminary criteria of interest for the user. CellRaft Gating can be performed in

Page 63 of 102 CellRaft AIR™ System User Manual one of two modes: 1) Single Cells mode, in which CellRafts are detected that contain a single cell based on a nuclear stain; and 2) Intensity Thresholds mode, in which CellRafts are filtered based on fluorescence intensities and the number of cells evident in the brightfield images. CellRafts that satisfy the given criteria are passed along the pipeline to the next step in the process – Marker Gating (see section 6.9.2.2) – and are available to select for isolation.

In Single Cells mode, images acquired during the Full Array Scan from the fluorescence Cell Counting Channel selected during Imaging Setup of the Full Array Scan (see section 6.7.2) are processed to count the number of cells resident on each CellRaft. For accurate cell counting, the fluorophore imaged in the Cell Counting Channel should correspond to a fluorescence signal that reveals a largely nuclear localization pattern. Please refer to Table 2 in section 4.3.3 regarding appropriate fluorophores to be used as the Cell Counting signal.

In Intensity Thresholds mode, images acquired during the Full Array Scan for all imaging channels are processed to quantify the fluorescence intensities and the number of cells present in the brightfield images for each CellRaft. Minimum and maximum thresholds can be set by the user on each of the imaging channels to gate the CellRafts that meet the desired characteristics.

Figure 29: Single Cells mode of CellRaft Gating screen in Cytometric Image Analysis mode.

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The controls in the CellRaft Gating screen (Figures 29 and 31) are designed to allow the user to review the accuracy with which the software gates the CellRafts based on the user specifications, whether it is identification of single-cell CellRafts or filtering based on fluorescence intensity, as well as modify the thresholds for the gating metrics. In Single Cells mode, the user can also apply rudimentary filters to the CellRaft Gating population based on confidence in the cell count and the intensity and size of the nuclear signal.

NOTE: If using a multi-reservoir (e.g. Quad or HexaQuad) CytoSort Array whose Full Array Scan imaging parameters were identical for all reservoirs, CellRaft Gating can be performed across all reservoirs or specific to each reservoir. If using a multi-reservoir CytoSort Array whose Full Array Scan imaging parameters differ between reservoirs, CellRaft Gating and Marker Gating must be performed specifically for each reservoir.

6.9.2.1. Single Cells mode of CellRaft Gating

Functionality of each of the controls in Single Cells mode (Figure 29) of CellRaft Gating is outlined below:

Primary Channel: one of the three fluorescence imaging channels is selected for use as the Cell Counting Channel. The selection defaults to the channel specified during Imaging Setup of the Full Array Scan, but can be changed during CellRaft Gating. Changing the Cell Counting Channel requires re-processing of the Full Array Scan data based on the new channel selection.

Primary Channel Histogram: shows the distribution of fluorescence intensities within the Cell Counting Channel across all fields of view within the Full Array Scan. A slider is used to position an intensity threshold for nuclear fluorescence detection. Adjusting the slider modifies the appearance of the Primary Channel Mask Image in real time; however, the new threshold is not applied to all array scan images to update the single-cell population until the “Calculation Required” button is selected.

Calculation Required Button: If the user changes the Cell Counting Channel or alters the intensity threshold on the Primary Channel Histogram, a blue “Calculation Required” button and a blue “X” button will appear (Figure 30). Selecting the blue “X” button discards any changes to the Cell Figure 30: “Calculation Required” button in CellRaft Gating screen of Cytometric Image Analysis mode.

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Counting Channel or intensity threshold. Selecting the “Calculation Required” button will prompt the software to re-process the Cell Counting Channel images from the Full Array Scan using the new channel and threshold to update the single-cell CellRaft population (a process which takes 30 seconds for single- and quad-reservoir arrays and several minutes for HexaQuads). Once complete, a new population of single-cell CellRafts will be available for display and the Cell Counting Statistics will refresh.

Cell Counting Statistics: Several statistics from the CellRaft Gating analysis are displayed below the Primary Channel Histogram:

Potential CellRafts: The software automatically analyzes the Full Array Scan imaging data to identify the CellRafts within the CytoSort Array whose fluorescence intensity within the Cell Counting Channel is above the intensity threshold. If the software detects an abnormality in the array (e.g. debris, defect), the affected CellRafts will be excluded from the analysis.

Total Cell Candidates Detected: This represents the total number of distinct fluorescent objects detected across all Potential CellRafts in the Cell Counting Channel. This can be used as a surrogate for total cells seeded but often small objects or debris which exhibit autofluorescence can be detected as objects. Further, an artificially high intensity threshold can underestimate the number of objects present. This statistic should not be viewed as an alternative to a hemocytometer or other cell counting device.

Single Cell CellRaft Candidates Available for Gating: This represents the number of CellRafts identified by the system which are likely to contain a single cell based on the nuclear signal threshold in the Cell Counting Channel and the filter settings described below. This population is transferred to the Marker Gating step for further analysis and represents the population available from which the user can select CellRafts for isolation.

Full Array Scan Channel Images: Images from an individual CellRaft corresponding to the brightfield channel, the three fluorescence channels, and a fluorescence composite are displayed. Only CellRafts corresponding to the “Single Cell CellRaft Candidates Available for Gating” are available for display.

Primary Channel Mask Image: The “mask” image provides a depiction of the analysis performed by the software to identify the number of distinct fluorescent “objects” within the Cell Counting Channel image of the currently displayed CellRaft. Adjustments to the intensity threshold on the histogram are reflected in the appearance of the mask in real time. Given that only CellRafts deemed to contain a single cell are available for display, the mask will initially depict a single white object. Overlapping nuclei can cause the system to erroneously count two or more cells as a single cell. Increasing the intensity threshold may allow separation of the nuclei, but could also exclude nuclei with weak fluorescence signals in the displayed or other CellRafts.

View Next or Previous CellRaft: These buttons can be used to cycle the Sample Images through the population of CellRafts which the software has predicted contain a single cell. Cycling through the

Page 66 of 102 CellRaft AIR™ System User Manual population can give the user a comprehensive view of overall image quality and the accuracy of single-cell detection at the current intensity threshold and filter settings.

Thumbs up/down and Star Ratings: These controls can be used to assign ratings to the current CellRaft to more easily track the CellRaft over the course of an experiment using the CellRaft Organizer as described in section 6.10. Assigning a thumbs-down rating immediately excludes the CellRaft from the remainder of the Cytometric Image Analysis. WARNING: Once a CellRaft is excluded, the only method to remove the thumbs-down rating is through the CellRaft Organizer, requiring exit from Cytometric Image Analysis mode.

Contrast Adjustment Histograms: Histograms are displayed below each Sample Image with sliders that can be manipulated to improve the image contrast for improved visualization. Note that these settings impact only the image display within the interface and will not be reproduced in the raw CellRaft images saved as part of the experimental report. The images saved with the report can be manipulated at the user’s discretion after the experiment.

Filter Settings: Several filters are available to screen detected single-cell CellRafts from further analysis. Modification of the filter settings only impact the “Single Cell CellRaft Candidates Available for Gating” statistic reported above.

Confidence: The algorithms used to threshold the Cell Counting Channel images and count the number of fluorescence objects within each CellRaft produce a confidence level that a given CellRaft contains a single cell. The confidence levels are grouped into bins of High, Medium and Low. Sliders on the filter control allow the user to set the range of confidence levels to include in the population for display, further analysis, and selection for isolation.

Higher degrees of confidence are recommended to accelerate the overall workflow for isolating single cells, provided sufficient numbers of single cells are available within those classification bins. Conversely, if a low proportion of cells within the overall single-cell population are expected to be of interest, including the Low confidence bin may be required.

Maximum Intensity: A range can be applied to the maximum pixel intensity within the detected fluorescent object (i.e. nucleus) to eliminate CellRafts whose signal intensities do not align with the user’s desired nuclear population. The axis and threshold values correspond to the “Max Int” statistic reported for the currently displayed CellRaft below the Filter Settings controls.

Size: A range can be applied to the overall size of the detected fluorescent object (i.e. nucleus) to eliminate CellRafts with single fluorescent objects that do not correspond in size to the user’s desired nuclear population. The axis and threshold values correspond to the “Pixels” statistic reported for the currently displayed CellRaft below the Filter Settings controls.

Marker Gating tab: Once the user is satisfied with the CellRaft Gating parameters, clicking on the “Marker Gating” tab at the top of the screen moves the Cytometric Image Analysis pipeline to the

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Marker Gating screen and passes all CellRafts included in the “Single Cell CellRaft Candidates Available for Gating” population to the Marker Gating step.

Return to Home tab: Clicking the “Return to Home” tab at the top of the screen returns to the Off the AIR module Home screen; however, returning to Home will clear any CellRaft Gating selections.

6.9.2.2. Intensity Thresholds mode of CellRaft Gating

Functionality of each of the controls in Intensity Thresholds mode (Figure 31) of CellRaft Gating is outlined below:

Fluorescence Channel Histograms: Show the distribution of fluorescence intensities within the each of the acquired fluorescence imaging channels across all fields of view within the Full Array Scan. Two sliders are used to position minimum and maximum intensity thresholds for fluorescence intensity filtering. Adjusting the sliders filters the CellRaft population and updates the CellRaft statistics in real time.

Figure 31: Intensity Thresholds mode of CellRaft Gating screen in Cytometric Image Analysis mode.

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Brightfield Areas of Interest: Shows the distribution of the estimated number of cells present on each CellRaft within the brightfield images. The numbers of cells are divided into four bins: zero, one(+) – high confidence of one cell, one(-) – low confidence that it is only one cell, and two or more. Two sliders are used to position minimum and maximum cell number thresholds for brightfield cell detection filtering. Adjusting the sliders filters the CellRaft population and updates the CellRaft statistics in real time.

Exclude Fiducials checkbox: If selected, all CellRafts that contain alphanumeric or “dot” fiducial markers are excluded from the CellRaft gating population.

CellRaft Statistics: Several statistics from the CellRaft Gating analysis are displayed below the Primary Channel Histogram:

Potential Rafts: This represents the nominal number of CellRafts within the given CytoSort Array format and based on whether fiducial CellRafts are included or excluded.

Rafts Available for Gating: This represents the number of CellRafts identified by the system whose fluorescence intensities and brightfield areas of interest satisfy the user-specified threshold within each of the controls. If the software detects an abnormality in the array (e.g. debris, defect), the affected CellRafts will be excluded from the analysis. This population is transferred to the Marker Gating step for further analysis and represents the population available from which the user can select CellRafts for isolation.

Full Array Scan Channel Images: Images from an individual CellRaft corresponding to the brightfield channel, the three fluorescence channels, and a fluorescence composite are displayed. Only CellRafts corresponding to the “Rafts Available for Gating” are available for display.

View Next or Previous CellRaft: These buttons can be used to cycle the Sample Images through the population of CellRafts which satisfy the user-specified threshold within each control. Cycling through the population can give the user a comprehensive view of overall image quality and image CellRaft characteristics that adhere to the current threshold settings.

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Marker Gating tab: Once the user is satisfied with the CellRaft Gating parameters, clicking on the “Marker Gating” tab at the top of the screen moves the Cytometric Image Analysis pipeline to the Marker Gating screen and passes all CellRafts included in the “Single Cell CellRaft Candidates Available for Gating” population to the Marker Gating step.

Return to Home tab: Clicking the “Return to Home” tab at the top of the screen returns to the Off the AIR module Home screen; however, returning to Home will clear any CellRaft Gating selections.

6.9.3. Marker Gating in Cytometric Image Analysis mode

The Marker Gating screen (Figure 32) is designed to allow the user to delineate sub-populations within the CellRaft Gated population based on a variety of CellRaft analysis metrics, including fluorescence intensity, number of cells as detected in brightfield, percent confluency in the CellRaft, and changes in these metrics between timepoints (i.e. full array scans). The metrics from the “Single Cell CellRaft Candidates Available for Gating” population (see section 6.9.2.1) or “Rafts Available for Gating” population (see section 6.9.2.2) are charted on an X-Y scatterplot, with each CellRaft represented by a

Figure 32: Marker Gating screen in Cytometric Image Analysis mode.

Page 70 of 102 CellRaft AIR™ System User Manual single yellow circle. Any of the CellRaft analysis metrics can be selected for the X and Y axes. A series of “gates” can be drawn on the histogram to create sub-populations of cells.

NOTE: If using a multi-reservoir (e.g. Quad or HexaQuad) CytoSort Array whose Full Array Scan imaging parameters were identical for all reservoirs, Marker Gating can be performed across all reservoirs or specific to each reservoir. If using a multi-reservoir CytoSort Figure 33: CellRaft analysis metrics during Marker Gating. Array whose Full Array Scan imaging parameters differ between reservoirs, Cell Gating and Marker Gating must be performed specifically for each reservoir.

Functionality of each of the controls in the Marker Gating screen is outlined below:

Established Marker Gates: These icons represent reduced-size versions of the Active Marker Gate Histogram for the sub-population defined by each marker gate. Each icon can be clicked to select the gate as the Active Marker Gate and show the gate population in the Active Marker Gate Histogram. An Established Marker Gate can also be deleted by selecting the red “X” in the upper- right corner of its icon. Initially, the only Established Marker Gate (labeled gate A) that is displayed corresponds to the entire CellRaft population calculated during CellRaft Gating.

Active Marker Gate Scatterplot: Each yellow dot on the histogram represents a single CellRaft within the Active Marker Gate. The initial Active Marker Gate (labeled gate A) corresponds to the entire CellRaft population calculated during CellRaft Gating. New Marker Gates can be created on the histogram and by selecting the circular arrow at the end of each axis label, the axes can be adjusted to correspond to any of the following CellRaft metrics (Figure 33): 1. Blue/Green/Red RFU – relative fluorescence intensity of the CellRaft within the specified imaging channel 2. Brightfield AOIs (Areas of Interest) – estimated number of cells present on each CellRaft within the brightfield images. This metric is assigned a value [0, 1, 1.5, 2, 3, 4, 5]: a. 0 if there are no CellRaft areas showing brightfield contrast due to lack of cells b. 1 if there is one area of brightfield contrast and high confidence that the signal comprises a single cell (corresponding to 1(+) designation during CellRaft Gating) c. 1.5 if there is one area of brightfield contrast but low confidence that the signal comprises a single cell (corresponding to 1(-) designation during CellRaft Gating) d. 2, 3, or 4 corresponding to that many distinct areas showing brightfield contrast e. 5 if there are five or more distinct areas showing brightfield contrast. 3. Brightfield Confluency – estimated proportion of CellRaft area showing brightfield contrast due to presence of cells 4. Blue/Green/Red RFU – change in relative fluorescence intensity of the CellRaft within the specified imaging channel between two timepoints

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5. Brightfield AOIs – change in brightfield areas of interest in the CellRaft between two timepoints 6. Brightfield Confluency – change in brightfield confluency within the CellRaft between two timepoints. If multiple full array scans have been included in the Cytometric Image Analysis, sliders along the histogram axes allow the user to specify the full arrays scans (i.e. timepoints) to use to calculate the specified metric. Metrics #1 – 3 require a single timepoint, while metrics #4 – 6 require two timepoints to be specified. A given metric can be used for both axes but set to different timepoints.

Show Rafts in Selected Gate checkbox: During the marker gating process, the user can check the “Show Rafts in Selected Gate” checkbox to overlay an Image Review window to review the images of a randomly selected CellRafts from the Active Marker Gate population (Figure 34) to evaluate the settings for the current marker gate before moving to Plate Mapping. CellRaft images are shown in all imaging channels collected during the Full Array Scan. Within the Image Review window, the user can use the arrow buttons to cycle the positions of the various imaging channels. The largest image position in the window contains a pixel histogram with controls to adjust the contrast within the image for better visualization; however, adjustment does not impact the underlying raw imaging data. If multiple full array scans have been included in the Cytometric Image Figure 34: The Image Review window during Marker Gating Analysis, controls allow the user to cycle within Cytometric Image Analysis mode. between the full array scans for the currently displayed CellRaft. De-selecting the checkbox dismisses the Image Review window.

New Marker Gate Menu: To set up a new gate within the Active Marker Gate Histogram, one of the New Marker Gate Menu icons is clicked. There are multiple options for gate geometries: quadrant, ellipse, line and polygon. Once a gate is established (as described in “Generating a New Marker Gate” below), it is given an appropriate identifier and displayed in the Established Marker Gates.

Active Marker Gate Identifier (ID): Each Marker Gate receives a unique identifier when it is created (Figure 35). Gates are labeled alphabetically in the order in which they were established for each reservoir, with gate A representing all CellRafts identified during CellRaft Gating. This letter is followed by a 4-character code comprising: the Figure 35: Marker gate naming letter representing the “parent” gate on which the gate was convention in Cytometric Image Analysis mode.

Page 72 of 102 CellRaft AIR™ System User Manual drawn, a hyphen, a letter representing the type of gate (L for line; Q for quadrant; E for ellipse; P for polygon) and a number in sequential order for that type of gate within the parent gate. The nomenclature is further described in “Generating a New Marker Gate” below. A sample figure of the identifier convention is found in. NOTE: If using a multi-reservoir CytoSort Array, all gate identifiers are preceded by the reservoir (A1-, A2-, B1-, B2-, ALL-) to which the gates correspond.

CytoSort Array Map: Each yellow dot on the schematic shows the position on the CytoSort Array of a single CellRaft within the Active Marker Gate. A label above the map indicates the number of CellRafts contained within the gate population.

Generating a New Marker Gate

To establish a new marker gate, a parent marker gate should be selected as the Active Marker Gate for display in the Active Marker Gate Histogram. Gate A will be initially available and represents all CellRafts identified during CellRaft Gating. Only CellRafts within the Active Marker Gate (i.e. the parent gate) will be included in the new marker gate population.

A marker gate geometry is then selected from the New Marker Gate Menu. The marker geometry can be drawn over the existing gate population displayed in the Active Marker Gate Histogram. Specifics for gate drawing are provided here:

Quadrant: A single click creates the intersection between a vertical line and a horizontal line, creating four potential sub-populations. Any or all of the four quadrants can be double-clicked to generate new marker gates. The naming of each quadrant is as follows: Q1 = Top Right; Q2 = Top Left; Q3 = Bottom Left; Q4 = Bottom Right. If a quadrant geometry is drawn on a parent gate, no other geometries can be drawn within that parent gate.

Line: Two clicks are required to create a line. The software will extend an infinite line in both directions through the two click positions, creating two potential sub-populations. Either or both sides of the line can be double-clicked to generate new marker gates. The side of the line containing the top-left corner of the parent gate is labeled L1 for the marker gate naming convention, while the other side of the line is labeled as L2. If a line geometry is drawn on a parent gate, no other geometries can be drawn within that parent gate.

Ellipse: Three clicks are required to create an ellipse. The first indicates the center of the ellipse. The second defines one radius and the rotation of the ellipse. The third click defines the other radius. The ellipse geometry creates a single potential sub-population within the ellipse. Double- clicking inside the ellipse generates a new marker gate. Multiple ellipse and polygon geometries can be drawn on a single parent gate. Elliptical gates are numbered (E1, E2, E3) according to the order of their generation from a given parent gate.

Polygon: Multiple clicks generate the corners of a polygon with the sides drawn between them. As many vertices as needed are permitted provided the sides do not cross one another. Clicking on the first corner closes and defines the polygon, creating one potential sub-population within

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the polygon. Double-clicking inside the polygon generates a new marker gate. Multiple ellipse and polygon geometries can be drawn on a single parent gate. Polygon gates are numbered (P1, P2, P3) according to the order of their generation from a given parent gate.

Promoting a Marker Gate: Since a given Marker Gate population may be an intermediary to a more refined CellRaft population, the user is able to “promote” Marker Gates to indicate to the software true sub-populations of interest. Promoting a Marker Gate makes it available for several purposes: 1. CellRaft members of the population will be available during Plate Mapping for selection for isolation; 2. The Marker Gate will be included in the Marker Gates list on the Off the AIR Home screen; 3. The Marker Gate will be included in the filter list in the CellRaft Organizer; and 4. The Marker Gate will be included in the gate list under the Report Generation tab of the Off the AIR module. To promote a Marker Gate population, select it from the Established Marker Gates as the Active Marker Gate for display in the Active Marker Gate Histogram; then, left- click on the Active Marker Gate ID above the CytoSort Array Map. The ID will change to an edit box in which the user can specify the name of the marker gate population and will include a “Promote” button to promote the marker gate (Figure 36). After promotion, a green checkmark will appear Figure 36: Promoting and naming a Marker Gate. within the icon of the Established Marker Gates panel.

CellRaft Gating tab: If the user wishes to revise the CellRaft Gating parameters, clicking on the “CellRaft Gating” tab at the top of the screen returns the Cytometric Image Analysis pipeline to the CellRaft Gating screen. WARNING: Reverting to the CellRaft Gating screen clears any Marker Gates already created (but retains the promoted Marker Gates in the database record).

Plate Mapping tab: Once the user is satisfied with the Marker Gates, clicking on the “Plate Mapping” tab at the top of the screen moves the Cytometric Image Analysis pipeline to the Plate Mapping screen where CellRafts can be selected from the promoted Marker Gate populations for isolation.

Return to Home tab: Clicking the “Return to Home” tab at the top of the screen returns to the Off the AIR module Home screen; however, returning to Home will clear any CellRaft Gating and Marker Gating selections (but retain the promoted Marker Gates in the database record).

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Figure 37: Plate Mapping screen in Cytometric Image Analysis mode.

6.9.4. CellRaft Selection for Isolation in Cytometric Image Analysis mode

The Plate Mapping screen (Figure 37) is designed to allow the user to randomly or explicitly select individual CellRafts from the promoted Marker Gate populations for isolation and assignment to locations within the collection plate.

The Plate Mapping screen contains a drop-down menu that lists the marker gate populations promoted from the Marker Gating screen. Similar to the Marker Gating screen, it also displays a two-dimensional fluorescence intensity scatterplot and the relative locations on the CytoSort Array of the CellRafts within the Active Marker Gate. The schematic of the collection plate is used to assign a selected CellRaft to a given well within the collection plate and display those assignments.

Functionality of each of the controls in the Plate Mapping screen is outlined below:

Active Marker Gate selection: The Active Marker Gate is identified at the top of the Plate Mapping screen using the name assigned by the user. Left-clicking the circular arrow to the left of the gate name drops down a list of Marker Gates promoted by the user in the Marker Gating screen. NOTE: only the Marker Gates promoted during the current session of Cytometric Image Analysis mode are

Page 75 of 102 CellRaft AIR™ System User Manual included. Selecting a Marker Gate population from the list selects that gate as the Active Marker Gate. The Active Gate Histogram and CytoSort Array schematic update to indicate the cytometric analysis metrics and locations for the CellRafts within the Active Gate population.

Sample Randomly Toggle: Selection of the Sample Randomly checkbox under the Active Marker Gate commands the software to select CellRafts randomly from the Active Gate population during plate mapping. Clicking on a well within the collection plate map assigns the randomly selected CellRaft to the well. Clicking on a row letter or column number assigns randomly selected CellRafts to all unoccupied wells within that row or column. With the checkbox deselected, the user must select CellRafts explicitly from the Active Gate histogram or CytoSort Array Map to fill the collection plate map.

Active Marker Gate Scatterplot: This area shows a two-dimensional scatterplot for the CellRafts within the Active Gate population for cytometric analysis metrics associated with the marker gate. CellRafts that have already been mapped for retrieval are colored red while those still available for selection are colored yellow. If the Sample Randomly checkbox is deselected for the Active Gate, a CellRaft can be selected by clicking on its yellow dot within the scatterplot. Upon selection, the CellRaft will turn blue. Upon assignment to a location within the plate map, the CellRaft will turn red.

CytoSort Array Map: This image depicts the relative locations of the CellRafts within the Active Gate population on a schematic of the CytoSort Array. CellRafts that have already been mapped for retrieval are colored red while those still available for selection are colored yellow. If the Sample Randomly checkbox is deselected for the Active Gate, a CellRaft can be selected by clicking on its yellow dot within the array map. Upon selection, the CellRaft will turn blue. Upon assignment to a location within the plate map, the CellRaft will turn red.

Collection Plate Map: Depicts a standard 96-position collection plate that can correspond to a 96- well culture plate, 96-well PCR plate, or 12, 8-tube 0.2mL PCR tube strips in the PCR Tube Adapter Plate in the AIR System Accessory Kit. • If the Sample Randomly checkbox is deselected for the Active Gate and the user has selected a CellRaft from the Active Gate Histogram or CytoSort Array Map, clicking on a well within the collection plate map assigns the selected CellRaft to that well. • If the Sample Randomly checkbox is selected for the Active Gate, clicking on a well within the collection plate map assigns a CellRaft randomly selected from that population to the well. Clicking on a row letter or column number assigns randomly selected CellRafts from the population to all unoccupied wells within that row or column. When a CellRaft is assigned to a given well: • The well in the collection plate schematic is filled with the CytoSort Array address of the CellRaft assigned to it. • The selected CellRaft is assigned to the Active Color Group, controlled by the colored circles in the lower-right of the screen. Clicking on the colored circle for a given color group selects that group as the Active Color Group; a bright green circle is used to indicate the selection.

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Show Images of CellRafts checkbox: During the plate mapping process, the user can select the Show Images of CellRafts checkbox to overlay an Image Review window to review the images of a selected CellRaft and confirm its image properties prior to selecting it for isolation (see Figure 38). CellRaft images are shown in all imaging channels collected during the Full Array Scan. Within the Image Review window, the user can use the arrow buttons to cycle the positions of the imaging channels. The largest image position in the window contains a pixel histogram with controls to adjust the contrast within the image for better visualization; however, adjustment does not impact the underlying raw imaging data. If multiple full array scans have been included in the Cytometric Image Analysis, controls allow the user to cycle between the full array scans for the currently displayed CellRaft. De-selecting the Show Images of CellRafts checkbox dismisses the Image Review window.

The Image Review window is invoked during CellRaft selection differently depending on the state of the Sample Randomly checkbox: 1. Without the Sample Randomly checkbox selected, the Image Review screen is invoked when the user selects a CellRaft from the Active Gate Histogram or the CytoSort Array Map. The screen is then dismissed when the CellRaft is assigned a location within the Collection Plate Map or by clicking the “Close” button (in place of the “New Raft” button in Figure 38). 2. With the Sample Randomly checkbox selected, the Image Review window is automatically invoked since the software independently selects a CellRaft from the Active Gate population. If the CellRaft is assigned a location within the Collection Plate Map, a new CellRaft is selected and the image display is updated. If instead the user does not wish to isolate the currently selected CellRaft, clicking the “New Raft” button selects a new CellRaft from the Active Gate Figure 39: The Image Review window to review the collection population. The only way to dismiss plate during Plate Mapping within Cytometric Image Analysis mode.

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the Image Review window in Sample Randomly mode is to deselect the “Show Images of CellRafts” checkbox.

Reviewing the Collection Plate: Clicking on an occupied well in the collection plate schematic also invokes the Image Review window to allow the user to inspect the images saved for the CellRaft assigned to that well (Figure 39). This Figure 40: Collection Plate Menu options during Plate capability can be used to quickly review the Mapping in Cytometric Image Analysis mode. images from randomly selected CellRafts that were assigned to the collection plate an entire row or column at a time. A second left-click in the collection well dismisses the Image Review window.

Modifying the Collection Plate: The assigned CellRaft locations within the collection plate map can also be modified by left-clicking on a given CellRaft position in the collection plate schematic and then left- clicking on any other position in the collection plate that is currently unoccupied. CellRafts may instead be deleted from the collection plate by left-clicking on their position in the plate, right-clicking on the same location to invoke the “trashcan” icon, then confirming deletion by right-clicking a second time.

Mapping multiple collection plates: Additional collection plates can be added for mapping by clicking on the Collection Plate Menu icon and selecting the green plus sign from the options (Figure 40). The user can toggle between plates using the left and right arrow keys.

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6.10. Tracking CellRafts using the CellRaft Organizer

The CellRaft Organizer screen is designed to allow the user to monitor CellRafts within the CytoSort Array throughout an experiment using the myriad parameters included in the database record. By combining many of the CellRaft review tools present in other mode screens, the goal is to allow the user to identify CellRafts of interest early in the experiment, then easily track those CellRafts as additional imaging information is acquired or additional cytometric analyses are performed, ultimately to determine which CellRafts to isolate and when to isolate them.

In the CellRaft Organizer, CellRafts are displayed in a table and can be filtered based on: • thumbs up/down and star ratings assigned from the any of the other mode screens or in the CellRaft Organizer • cytometric marker gate • collection plate number and color group • isolation status • time since review or modification in the CellRaft Organizer itself. CellRafts can be selected from the table to: • review images acquired during Full Array Scans or saved during User Navigation and Selection mode • change user-assigned ratings based on the most recent imaging information • assign locations in collection plates for isolation. Collection plate maps can even be reviewed and modified using functionality consistent with the User Navigation and Selection, User Review and Selection, and Cytometric Image Analysis (Plate Mapping) modes.

6.10.1. Entering the CellRaft Organizer screen

The CellRaft Organizer screen can be accessed from the Off the AIR software Home screen when either the “Full Array Scans” or “Marker Gating” tab is selected.

NOTE: Since the CellRaft Organizer screen is accessed through the Off the AIR software module, the analysis can be performed from any computer with the Off the AIR software installed and with access to the database record.

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Figure 41: CellRaft Organizer screen in Off the AIR software module.

6.10.2. CellRaft Organizer functionality

The CellRaft Organizer screen (Figure 41) is centered around a CellRaft table that allows the user to browse through a list of CellRafts within the CytoSort Array. The table is accompanied by images of the currently selected CellRaft, controls that allow the user to filter the list of CellRafts displayed in the table, and (if selected) a collection plate schematic to review, modify, or add to the collection plate maps within the database record.

Functionality of each of the controls in the CellRaft Organizer screen is outlined below:

CellRaft Table: Tabulated list of CellRafts in the CytoSort Array that satisfy the criteria in the Filter Builder. The CellRaft Organizer defaults to no filters set, so the initial list includes all CellRafts in the array. The columns in the table correspond to the number in the list, the array row and column addresses, the user-assigned thumbs up/down and star ratings, and collection plate parameters including plate number, plate well, color group, and isolation status.

Controls within the table include: • The user can change the currently selected CellRaft by left-clicking within the number, Row, or Col columns of the new CellRaft.

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• The user can select multiple CellRafts from the table by left-clicking and dragging within the table, but only within a currently visible group of CellRafts. During multi-selection, the images of the first CellRaft in the list are displayed. • The user can navigate through the list by using the standard controls on the table scrollbar. • The user can change or set the thumbs up/down and star ratings for the currently selected CellRaft(s) using the controls in the User column. Assigning a thumbs-down rating will exclude the CellRaft from Cytometric Image Analysis. To remove a star rating from a CellRaft, the user can apply a thumbs-down rating followed by a thumbs-up rating.

Filter Builder: Allows the user to filter the list of displayed CellRafts in the CellRaft Table based on a variety of parameters tracked in the database record. To add a filter, the user left-clicks on the green “+” then on “Click to Select Metric” caption. A drop-down menu provides the filter options:

• Stars: the user can filter CellRafts based on an operator (<, <=, =, >=, >) and a threshold number of stars. A lack of star rating is equivalent to zero stars. Clicking on the operator and the threshold invokes drop-down menus for each. • Show Thumbs Down Rafts: the user can filter CellRafts based on whether a thumbs down rating has been applied. A True setting will only display CellRafts assigned a thumbs down rating. A False setting will display all others. Clicking on the True/False designation invokes a drop-down menu. • Marker Gate: the user can filter CellRafts based on their membership within a promoted cytometric marker gate. This filter is inclusive only, meaning that gate membership cannot be used to exclude CellRafts from the table. Clicking on the gate name invokes a drop-down menu of promoted cytometric gates.

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• Collection Plate: the user can filter CellRafts based on their mapping to a given collection plate number. This filter is inclusive only, meaning that collection plate mapping cannot be used to exclude CellRafts from the table. Clicking on the collection plate description invokes a drop-down menu of mapped

collection plates. • Collection Plate Status: the user can filter CellRafts based on isolation status. Clicking on the collection plate description invokes a drop- down menu of isolation states: o Mapped = assigned a position in a collection plate map o Not Mapped = not assigned a position in

a collection plate map o Isolated = released and transferred according to section 6.11 o Not Released = isolation attempted but failed to release from CytoSort Array o Not Collected = isolation attempted but failed to collect by the magnetic wand • Collection Plate Group: the user can filter

CellRafts based on their membership to a given color group in the collection plate maps. This filter is inclusive only, meaning that collection plate color group cannot be used to exclude CellRafts from the table. Clicking on the color group description invokes a drop-down menu of color group names. • Reservoir: the user can filter CellRafts based on

their reservoir within a multi-reservoir array (e.g. Quad or HexaQuad). Clicking on the reservoir description invokes a drop-down menu of reservoirs within the array. • Hours Since Status Modified: the user can filter CellRafts based on the time since the state of any of the aforementioned parameters has changed. The user can select an operator (<= or >=) and enter a (integer) number of hours in the edit box. Clicking on the operator invokes a drop-down menu. • Hours Since Viewed in Organizer: the user can filter CellRafts based on the time since the user last viewed the CellRaft in the CellRaft Organizer. The user can select an operator (<= or

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CellRaft AIR™ System User Manual

>=) and enter a (integer) number of hours in the edit box. Clicking on the operator invokes a drop-down menu. Multiple filters can be applied, with only the CellRafts that satisfy ALL filters (“AND” condition) displayed in the table.

CellRaft Images: Images from the currently selected CellRaft corresponding to the brightfield channel (at the top of the CellRaft and the top of the microwell wall), the three fluorescence channels, and a fluorescence composite are displayed. Images correspond either to Full Array Scan images (if acquired) or images saved during User Navigation mode when a CellRaft is assigned a position in a collection plate map. If multiple full array scans acquired from the CytoSort Array, controls allow the user to cycle between the full array scans for the currently displayed CellRaft.

Contrast Adjustment Histograms: Histograms are displayed below each CellRaft image with sliders that can be manipulated to improve the image contrast for improved visualization. Note that these settings impact only the image display within the interface and will not be reproduced in the raw CellRaft images saved as part of the experimental report. The images saved with the report can be manipulated at the user’s discretion after the experiment.

Show Collection Plate checkbox: Selection of this checkbox removes the right two CellRaft images and displays the Collection Plate Map for review and modification of the collection plate maps in the database record.

Collection Plate Map: Depicts a standard 96-position collection plate that can correspond to a 96- well culture plate, 96-well PCR plate, or 12, 8-tube 0.2mL PCR tube strips in the PCR Tube Adapter Plate in the AIR System Accessory Kit. Clicking on an unoccupied well within the collection plate map assigns the currently selected CellRaft to that well. If multiple CellRafts are selected, the CellRafts are assigned to the selected well and subsequent unoccupied wells along the selected row and subsequent (i.e. higher number) rows. When a CellRaft is assigned to a given well: • The well in the collection plate schematic is filled with the CytoSort Array address of the CellRaft assigned to it. • The selected CellRaft is assigned to the Active Color Group, controlled by the colored circles below the collection plate map. Clicking on the colored circle for a given color group selects that group as the Active Color Group; a bright green circle is used to indicate the selection.

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NOTE: Reviewing the Collection Plate as described in other modes is not supported in the CellRaft Organizer screen. As such, clicking on an occupied well in the collection plate schematic DOES NOT invoke display of the images saved for the CellRaft assigned to that well.

Modifying the Collection Plate: The assigned CellRaft locations within the collection plate map can be modified by left-clicking on a given CellRaft position in the collection plate schematic and then left-clicking on any other position in the collection plate that is currently unoccupied. CellRafts may instead be deleted from the collection plate by left- clicking on their position in the plate, right- clicking on the same location to invoke the “trashcan” icon, then confirming deletion by right-clicking a second time.

Mapping multiple collection plates: Additional collection plates can be added Figure 42: Collection Plate Menu options within the CellRaft for mapping by clicking on the Collection Organizer screen. Plate Menu icon and selecting the green plus sign from the options (Figure 42). The user can toggle between plates using the left and right arrow keys.

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6.11. Release and Transfer of CellRafts Selected for Isolation

Regardless of the modes of CellRaft imaging or selection, the AIR System workflow culminates with the CellRaft isolation process, during which CellRafts are one-by-one released from the CytoSort Array, collected by the magnetic collection wand, and deposited into the collection plate.

NOTE: CellRaft isolation is only compatible with the AIR software, not with Off the AIR.

6.11.1. Initiating CellRaft Isolation

From the Home screen in the AIR software with the Isolation Plate Maps tab selected, the software will list the collection plate maps created by the user and available for isolation. The radio buttons on the left end of the rows allows the user to select a collection plate for display and modification.

Selecting the checkbox next to “Edit Mode” allows the user to:

• Use the Collection Plate Type drop-down menu to select from a list of compatible plate types for use during CellRaft isolation. The plate type can be selected independently for each plate map in the list.

• Enable the “A1 rinse” option – clicking the sprinkler head at the right end of the row toggles between the enabled and disabled states. If water is coming out of the sprinkler head, the A1 rinse option is enabled and the AIR System will immerse the tip of the magnetic collection wand in the A1 well of the collection plate – which can be filled with buffer, ethanol, or some other rinsing solution – before the first isolation and after every isolation to prevent cross- contamination between collection wells.

NOTE: If a CellRaft is mapped to the A1 position within the collection plate and the A1 rinse option is enabled, the CellRaft mapped to position A1 will not be isolated.

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Once satisfied with the collection plate settings, de-selecting the box next to “Edit Mode” and clicking the “Isolate” button at the right end of the row initiates the CellRaft isolation process for the selected collection plate.

The software will prompt the user to:

• Place the CytoSort Array associated with the database record into the CytoSort Array Adapter Plate and load them into the left position of the AIR System stage. The CytoSort Array can either be placed in the adapter plate before or after inserting the adapter plate into its deck position (see section 6.2.5). Guide railings in the stage well assure proper positioning of the CytoSort Array. The Adapter Plate should be inserted so the arrow on the plate points toward the interior of the system. • Place the appropriate collection plate into the right position of the AIR System stage. NOTE: Refer to sections 6.2.6 – 6.2.7 for recommendations regarding various collection plate geometries and Attachment 1 for validated collection plate brands and part numbers, as well as compatible collection fluids and minimum fluid volumes.

NOTE: PCR tube strips and PCR plates should always be mounted in the PCR Tube Adapter Plate provided with the AIR System Accessory Kit when loaded into the AIR System.

• Attach the appropriate magnetic collection wand for the given collection plate type. Clicking the “Go to Wand Access Position” button will move the collection wand attachment plate to a middle position within the door aperture for easier access.

NOTE: The magnetic collection wand should always be attached with the Figure 43: Cut-away of AIR System model showing magnetic wand attachment.

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compression key on the blue connector facing the front of the instrument (Figure 43). Simply insert the connector end of the wand vertically into the aperture in the bottom of the attachment plate until a click is heard.

To detach the wand from the attachment plate, simply squeeze the compression key on the blue connector and pull downward to slide the connector from the attachment plate.

The Standard Wand and PCR Tube Wand are also compatible with a standard ethanol or isopropanol wipe of the wand surface for sterilization. However, do NOT soak the Standard Wand tip in alcohol for extended periods as it can degrade the wand surface.

Upon hitting the “Continue” button, the AIR System will calibrate the position of the CytoSort Array for navigation during imaging.

6.11.2. Executing CellRaft Isolation

Once the array calibration process is complete, the Release and Transfer screen will appear (Figure 44). A list of CellRaft addresses selected for isolation is provided on the left side of the screen. A schematic of the CytoSort Array with highlighted locations of CellRafts to be isolated is also provided in the middle. Finally, a map of the collection plate is shown on the right.

Clicking the “Isolate” button initiates the release and transfer process. Its progress will be continuously updated, including an estimated time of completion. • Green highlighted CellRafts have already been successfully isolated by the system. • Yellow highlighted CellRafts are undergoing active isolation. The image in the lower left shows before and after brightfield images of the CellRaft targeted for isolation (red crosshairs). • Red highlighted CellRafts failed to be successfully isolated via incomplete release by the needle. • Orange highlighted CellRafts failed to be successfully isolated via failed collection by the magnetic wand. • Black highlighted CellRafts failed segmentation and therefore could not be isolated, usually due to debris, a defect in the microwell array, or a redundancy in the imported Release and Transfer List.

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Figure 44: Release and Transfer screen during CellRaft isolation.

During the CellRaft isolation process, the AIR System software acquires and displays a brightfield image of the target CellRaft before and after attempted isolation to detect whether CellRaft release by the needle or collection by the magnetic wand failed during the isolation process. If release and collection were successful as determined through the images, the magnetic wand will move to the collection plate to deposit the CellRaft. If either release or collection failed during the first attempt, the system will attempt release and collection a second time. Regardless of the success during the second attempt, the magnetic wand will move to the collection plate to deposit the CellRaft.

NOTE: The release and transfer process can be paused at any time; however, if the process is paused and the “Return to Home” tab is clicked then the isolation process will be terminated and the Home screen will indicate that a subset of the CellRafts were isolated from the collection plate.

Once the CellRaft isolation is complete for the selected collection plate, the software will automatically return the microscope stage to the loading position, return the display to the Isolation Plate Maps tab of the AIR Home screen, and unlock the door to allow the user to unload the collection plate from the system. The “Access Wand” button below the “Edit Mode” checkbox can be used to move the collection wand attachment plate to a middle position within the door aperture for easier access to remove the collection wand. To detach the wand from the attachment plate, simply squeeze the compression key

Page 88 of 102 CellRaft AIR™ System User Manual on the blue connector and pull downward to slide the connector from the attachment plate, as described above.

6.11.3. Confirming Isolation of CellRafts in the Collection Plate

While the software is reasonably accurate in its detection of isolation failures, false failure reports have been observed and CellRaft deposition in the collection plate is not confirmed by the AIR System. Therefore, visual confirmation of the presence of a single CellRaft within each collection well is recommended. Successful deposit can be confirmed by examining the 96-well plate or PCR tubes under a microscope with modest magnification (2X-4X magnification dissection scope). A small orange square should be visible (the CellRaft) in the collection volume (Figure 45). Even if the software indicates that a given CellRaft was not released or not collected during CellRaft isolation, if a single CellRaft is present in the collection vessel, it is >99% certain to be the CellRaft intended for that position.

Figure 45: Example images of CellRaft deposition into a 96-well plate (left) and PCR strip tubes (right).

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6.12. Import a list of CellRaft Addresses for Isolation

If the user wishes to select CellRafts for isolation using an external microscope or some other means, the CellRaft AIR System software is capable of importing a list of CytoSort Array indices and collection plate locations for automated release and transfer with the instrument. Examining the CytoSort Array on a separate microscope may allow the use of imaging parameters not available on the AIR System.

The array indices and plate locations must be entered into a Microsoft Excel® template (Plate Map Template.xlsx) provided with the software which can then be directly imported for isolation of the listed CellRafts. The CellRaft address format is described in the CytoSort Array User Manual. Figure 46 depicts the format in which CellRaft Addresses should be entered into the Plate Map Template file prior to importing. The CytoSort Array row and column numbers should be entered into the orange shaded boxes within the template file. The collection plate rows and columns are Figure 46: Plate Map Template file used to import a list of arranged in row-major order, but can be re- CellRaft addresses for isolation with the AIR System. arranged in any order.

NOTE: Do NOT change the name of the “Plate Map” worksheet in the template file, as that worksheet is the map that is loaded by the AIR software; however, the Excel template file can be saved in a unique location and with a unique name.

To import the list of addresses into an Isolation Plate Map, the user should create a new database record according to section 6.4.2. From the AIR software module Home screen, clicking the “Off the AIR” button will move to the Home screen for the Off the AIR software. With the “Full Array Scans” tab selected, clicking the “User Selection” button will launch the Imaging screen.

In the top-left corner of the Imaging screen, the type of CytoSort Array will be indicated. Clicking the “Import Plate Map” button will launch a standard Microsoft Windows® “Open” dialog box.

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Within the dialog box, the user should select the Excel template file containing the CellRaft addresses to be isolated.

The CellRaft addresses will populate the collection plate. The addresses may be overwritten by re-clicking the “Import Plate Map” button and selecting a different template file.

As in other modes, the Collection Plate Menu in the bottom-right corner of the Imaging screen allows the user to add a new collection plate (green plus) to populate from another Excel template file, clear the currently viewed plate (purple eraser), or delete an existing plate from the list (red trash can). WARNING: there is currently no confirmation or “undo” of the erase and delete actions.

Once all template files have been loaded, clicking the “Return to Home” tab at the top of the screen returns to the Off the AIR Home screen in Figure 14 with any mapped collection plates now included in the database record and listed under the “Isolation Plate Maps” tab, where they can be selected for isolation (see section 6.11).

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6.13. Experimental Report and Images

From the AIR and Off the AIR Home screens, the Report Generation tab in the Tabbed Console lists the full array scans, isolation plate maps, and promoted cytometric marker gates associated with the database record to date and allows the user to select them for export to an Microsoft Excel® report. Each item listed can be selected for inclusion in the report or deselected for exclusion. The “Toggle All” checkbox below the Tabbed Console can be used to select or deselect all options. Clicking the “Generate Report” button generates the report and replaces the button with a hyperlink to the directory containing the report and image data. Reports are located within an AirReports sub-directory created by the software within the directory containing the database record on the computer hard drive (C:\CMSData\DatabaseName\AirReports\) or external SSD (X:\CMSData\DatabaseName\AirReports). The sub-directory and Microsoft Excel® file names for generated reports contain the timestamp of their creation date to enable export of multiple report combinations from a single database record and multiple timepoints within the experimental workflow.

6.13.1. Microsoft Excel report

Each Microsoft Excel report currently contains three worksheets: “FullArrayScans,” “CollectionPlates,” and “MarkerGates” – corresponding to the three sets of information selectable from the Report Generation tab of the AIR System software.

The FullArrayScans worksheet (Figure 47) contains three blocks of information:

Consumable: Lists information regarding the CytoSort Array associated with the database record from which the report was generated, including the array description (provided by the user on the Home screen), the unique database ID (provided by the user), the type of CytoSort Array (e.g. 200x200 Single Reservoir), and the creation date of the database record.

Scan History: Lists all full array scans acquired from the CytoSort Array, including the scan description (provided by the user on the Home screen), whether the scan is included in the report, the time difference between the start of the full array scan relative to the start of the first scan acquired from the array, and the timestamps of the start and end of the full array scan.

Imaging Parameters: Lists the imaging parameters for all reservoirs, all full array scans included in the report, and all imaging

channels, including the scan description Figure 47: FullArrayScans tab from experimental report.

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(provided by the user on the Home screen), the CytoSort Array reservoir, the imaging channel, whether the imaging channel was acquired during the scan, the camera exposure, gain and binning parameters for that scan/reservoir/channel combination, and whether the imaging channel was selected as the Cell Counting Channel.

The CollectionPlates worksheet (Figure 48) contains two blocks of information:

Plate History: Lists all collection plates mapped within the database record, including the plate name (provided by the user on the Home screen), the collection plate type (e.g. 96-well flat bottom plate), whether the collection plate map is included in the report, time stamps for the start and end of CellRaft selection within the plate map, and timestamps for the start and end of CellRaft isolation from the plate map.

Figure 48: CollectionPlates tab from experimental report. Mapped Wells: Lists all collection plate wells within the collection plate maps included in the report that contain a CellRaft selection, including: • the plate name (provided by the user on the Home screen) • the well index from the collection plate (e.g. F4) • the row and column addresses of the CellRaft assigned to the collection well • the status of CellRaft isolation at the time of report generation (e.g. Selected, Isolated, Not Released, Not Collected, Not Segmented) • the marker gate (Cytometric Image Analysis mode) or color grouping (User Navigation and Selection mode and User Review and Selection mode) to which the CellRaft belongs • Hyperlinks to the imaging channel images (as described below).

The MarkerGates worksheet (Figure 49) contains two blocks of information:

Marker History: Lists all cytometric marker gates promoted within the database record, including the marker gate description (provided by the user during promotion or on the Home screen), whether the marker gate is included in the report, the time stamp for the promotion of the marker gate, the unique identifier path during cytometric analysis, the type of CellRaft Figure 49: MarkerGates tab from experimental report.

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Gating (Channel Root), the reservoir(s) with which the gate is associated (Gate Sector – a value of 0 indicates the entire array), and the metrics and limits for the x- and y-axes associated with the gate.

Gate Members: Lists all CellRafts contained in each promoted marker gate that is included in the report, including the marker gate description (provided by the user during promotion or on the Home screen) and the row and column addresses of the CellRaft.

6.13.2. Exported Images

Within the report sub-directory created by the software, the AIR System software creates another sub- directory entitled “images.” The image directory contains the brightfield and fluorescence images associated with each CellRaft selected for isolation from each collection plate map selected for inclusion in the report. From Full Array Scans, the brightfield and fluorescence imaging channels active during the full array scan are exported. In User Navigation and Selection mode, the most recently acquired images from the CellRaft are saved for export from the active imaging channels at the time of selection and placement in the collection plate schematic.

CellRaft images are saved as Tagged Image Format (TIF) files and can be imported into ImageJ or any other image analysis software for display or further analysis. The image file names follow the naming convention, RR[R]CC[C]X.tiff, where RR[R] corresponds to the row index of the CellRaft within the CytoSort Array (two-character alphanumeric for single- and quad-reservoir arrays, three digit numeric for HexaQuads), CC[C] corresponds to the column index of the CellRaft within the CytoSort Array, and X corresponds to the imaging channel – A for a brightfield image, B/G/R for a blue/green/red fluorescence image, respectively.

NOTE: While images of all CellRafts are saved along with the database record, the report generation capability of the AIR System software does not export individual images of CellRafts that were not selected for isolation.

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7. Troubleshooting

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7.1. Overview

Several topics are discussed here for trouble shooting. These include: 1. Testing power connection and replacing power cord 2. Testing computer connection and replacing USB 3.0 cables 3. High number of uncollected CellRafts reported after release and transfer operation

Any technical issues other than those provided above should be reported to Cell Microsystems at the contact information provided at the beginning of this manual.

7.2. Testing Power Connection

If the AIR System does not start upon pressing the round power button on the front panel, ensure that the power cord provided with the system is properly connected between a 110V/60Hz or 220V/50Hz outlet and the power input module on the rear panel of the system.

Contact Cell Microsystems technical support for additional troubleshooting steps or a replacement power cord.

7.3. Testing Computer Connection

If the AIR System software (AIR software module) is not able to initialize the instrument during initialization, ensure that the AIR instrument is powered on and that the USB 3.0 cables provided with the system are properly connected between USB 3.0 ports on the computer and the USB 3.0 ports on the rear panel of the system. Both cables must be attached to the computer and the AIR™ System for full connectivity.

Generally, the computer should be powered on first, then the instrument, then the software can be launched. Approaching start-up in this order will better ensure connectivity is achieved.

Contact Cell Microsystems technical support for additional troubleshooting steps or replacement USB cables.

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7.4. High number of CellRaft isolation failures

Based on all recommendations in the AIR System User Manual and CytoSort Array User Manual, CellRaft isolation efficiency is expected to be greater than 90%. Isolation failures are indicated when the collection well is highlighted in red or orange within the Release and Transfer screen (see section 6.11.2) or when zero or 2+ CellRafts are observed in a collection well during isolation confirmation of the collection plate (see section 6.11.3). If the CellRaft isolation failure rate exceeds 20%, double-check the following: • the fluid volume in the CytoSort Array at the time of isolation is 5mL for a single-reservoir array and 1mL per reservoir for a quad-reservoir array • the fluid in the CytoSort Array contains at least 10% protein (FBS or BSA) to reduce the surface tension of the media • the Standard Wand (if applicable) was soaked in buffer for 10 minutes prior to isolation • the glass section of the PCR Tube Wand (if applicable) is still attached to its black strain relief – a light tug on the tube to ensure that it is unable to slide in and out of the strain relief is sufficient • the collection plate being used is listed or is dimensionally identical to one of the plates listed in Appendix 1 – if compatibility is in question, contact Cell Microsystems technical support • the wells in the collection plate contain at least the minimum fluid volumes based on the collection plate type, as outlined in Appendix 1 • the fluid volumes in the collection plate are free from bubbles injected during pipetting • for collection into fluid volumes below 5L in PCR tubes, that the fluid is positioned at the tip of the tube – centrifugation of the tube strips or PCR plate may be required • all released but uncollected CellRafts are removed from the CytoSort Array reservoirs prior to isolation – removal can be achieved under an inspection microscope using one of the wands in the AIR System Accessory Kit • all collected but undeposited CellRafts are removed from the Standard Wand or PCR Tube Wand prior to isolation – a simple ethanol wipe of the wand tip is usually sufficient • the release needle is still well aligned – this can be confirmed by observation of the brightfield images during CellRaft isolation (as described in section 6.11). During the initial CellRaft release attempt, the puncture marks should be centered roughly in the upper-left and lower-right quadrants of the target CellRaft. If the puncture marks are not visible, extend outside the limits of the target CellRaft, or appear to drift over time, or if neighboring CellRafts are being collaterally released with the target CellRaft, contact Cell Microsystems technical support.

If additional troubleshooting is required contact Cell Microsystems technical support.

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8. Appendix 1 – Labware Compatibility

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8.1. Validated flat-bottom 96-well culture plates

The following flat-bottom 96-well culture plates have been validated for CellRaft isolation with the CellRaft AIR System using the Standard Wand.

Minimum Part Number Distributor Plate Description Volume VWR Tissue Culture Plate, Non-Treated, Sterilized, Non-Pyrogenic VWR 10861-562 100L 96 well, flat bottom, clear https://us.vwr.com/store/product/18962910/vwr-tissue-culture-plate-non-treated-sterilized-non-pyrogenic Thermo Scientific Nunc MicroWell 96-well Microplates Thermo 168055 100L Cell culture, high flange, without lid

https://www.thermofisher.com/order/catalog/product/168055?SID=srch-srp-168055 Nunc MicroWell 96-well Optical-Bottom Plates with Polymer Base Thermo 165306 100L White, cell culture

https://www.thermofisher.com/order/catalog/product/165306?SID=srch-hj-165306

8.2. Validated PCR tubes

The following 0.2mL PCR tube strips have been validated for CellRaft isolation with the CellRaft AIR System using the PCR Tube Wand and mounted in the PCR Tube Adapter Plate.

Part Minimum Distributor Plate Description Number Volume Sapphire PCR Tubes and Strips, Polypropylene, Greiner Bio-One VWR 75796-124 2.5L 0.2 mL, without cap, natural https://us.vwr.com/store/product/20823817/sapphire-pcr-tubes-and-strips-polypropylene-greiner-bio-one PCR Tube Strips and Caps, Axygen VWR 89080-524 2.5L 0.2 mL, flat cap (removable) https://us.vwr.com/store/product/8950946/pcr-tube-strips-and-caps-axygen

8.3. Validated PCR plates

The following 0.2mL semi-skirted PCR plates have been validated for CellRaft isolation with the CellRaft AIR System using the PCR Tube Wand and mounted in the PCR Tube Adapter Plate.

Minimum Part Number Distributor Plate Description Volume Fisher Eppendorf twin.tec 96 Well LoBind PCR Plates, semi-skirted E0030129504 2.5L https://www.fishersci.com/shop/products/eppendorf-twin-tec-96-lobind-pcr-plates-semi-skirted-clear/e0030129504 USA Scientific TempPlate semi-skirted 96-Well PCR plates, straight skirt, natural 14029200 2.5L https://www.usascientific.com/semi-skirted-96-well-PCR-plate-straight-skirt.aspx

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9. Appendix 2 – Fluorescent Dye Compatibility

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9.1. Fluorescent Dyes and Stains Compatible with the AIR System

The standard configuration of the CellRaft AIR System provides three fluorescence imaging channels denoted as BLUE, GREEN, and RED. The excitation and emission bands of the three imaging channels derive from a combination of components within the AIR System inverted microscope: • the excitation spectra of the LEDs within the fluorescence LED engine • the transmission spectra of the excitation bandpass filters within the fluorescence LED engine • the reflection/transmission spectrum of the triple-band dichroic filter in the fluorescence filter set • the transmission spectrum of the triple-band emission filter in the fluorescence filter set.

Table 1 (reproduced here for Channel Representative Dyes Excitation Emission convenience) and Figure 50 Blue Hoechst, SYTO 41 378-401 nm 412-453 nm provide technical data regarding Green FITC, Alexa488 460-490 nm 497-548 nm the spectral bands of the standard Red Texas-Red 559-591 nm 602-805 nm imaging channels on the AIR System to help assess compatibility Table 1: Excitation and emission specifications for the three fluorescence imaging channels enabled by the AIR™ System (standard configuration). of fluorescent dyes and stains. Specific dyes and staining protocols will require user validation on the AIR System to assess image quality.

BLUE Imaging GREEN Imaging RED Imaging Channel Channel Channel

Violet LED BLUE - Emission Cyan LED GREEN - Emission Yellow LED RED - Emission

Dichroic Filter Transmission

300 350 400 450 500 550 600 650 700 750 800 850 900 Wavelength (nm)

Figure 50: Excitation and emission spectra for each of the three channels enabled by the AIR™ System (standard configuration).

Custom LED and filter configurations are available on the AIR System – contact Cell Microsystems to inquire. Such configurations are implemented during AIR System manufacturing and are not designed to be modified by the user in the laboratory.

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