SY3200 Cell Sorter true innovation inside

Europe True Innovation The SY3200™ system is one of the most innovative and versatile Flow Cytometry Research platforms available in the market.

True innovation drives superior performance and versatility in the SY3200 system. From optics to electronics, revolutionary technology provides solutions that enable the complex applications of today and tomorrow.

Advancements in discovery of new markers and new dyes have improved the ability to identify more populations in a single assay. This has advanced the use of flow cytometry in an increasing number of applications as well as challenged instrumentation to continue to adapt to new parameters. The SY3200 has been designed to easily adapt to the ever increasing need for flexibility and sensitivity in new applications. Whereas some systems are merely evolutionary in design, the SY3200 brings revolutionary, innovative and cutting edge technology to elevate the science of flow cytometry.

The innovative and flexible laser delivery array allows for ultimate laser compatibility while the unique reflective collection optics ensure maximum fluoresence collection efficiency. Powerful, Sony designed electronics enable collection of high resolution data and ultra high speed cell sorting while maintaining superior sensitivity. Plug and Play PMTs in the Scalable PMT Array (SPA) module provide the ultimate flexibility in collection options while minimizing cost.

The SY3200 provides true innovation to deliver a comprehensive cell sorting solution that meets the challenges of the increasing complexity of research flow cytometry applications. Ultimate Configurability

The SY3200 platform is designed for configurability to adapt to the demanding needs of a variety of sorting labs. Unique to any system in flow cytometry, the SY3200 is expandable to a dual system layout, effectively enabling two systems in one. All configurations of the SY3200 can share the detector and electronics console to allow for expansion to a second independent system with a marginal increase in lab space.

This dual Highly Automated Parallel Sorting (HAPS) configuration of the SY3200 can effectively double your lab’s capacity with a single instrument platform. For example, you can easily half the time of long, rare event sort with a additional sorter setup. Alternatively, one module can be setup for routine immunophenotyping applications, while the second can be dedicated to special applications such as side population assay, basic GFP sorting or optimized for multiple fluorescent protein analysis and sorting.

Benchtop System The benchtop system is a compact ergonomic design that fits easily on a standard lab bench. This configuration is highlighted by accessibility to all system components to allow easy configuration of laser and detector setups.

Floor Standing System The floor standing system delivers a unique design for labs that are bench space limited. This configuration which includes an integrated workbench can be easily upgraded to a BSC.

Integrated Biosafety Cabinet

The integrated biosafety cabinet option is yet another truly innovative approach in design that is tailored to the unique requirements of a lab performing biohazardous or sterile sorting. Innovation in Biosafety

Since the introduction of the multi-channel Cell Sorting System in 2006, Sony Biotechnology Inc. has been the leader in providing cell sorters with a truly integrated Bio Safety Cabinet. The SY3200 BSC system is no exception, delivering a carefully engineered integrated Bio Safety Cabinet design that provides operator, sample and environmental protection while main- taining an ergonomic and functional workspace. The SY3200 BSC is integrated into a Baker biosafety cabinet thereby exposing only minimum system components inside the hood.

The SY3200 BSC provides a compact footprint with internal workspace of 48” width. Even with two HAPs modules installed, there is ample workspace for safe sample handling, pipetting functions and sample preparation.

Simple and Safe Biohazard Sorting The Integrated Biosafety configuration has been specially designed to complement the demanding requirements of working in a BSL 2 or 3 laboratory. Only the minimum system components are exposed within the cabinet. This allows for both integrity of a biosafe environment as well as easier maintenance. With the HAPS module or modules elevated above the biosafety cabinet work surface area, the SY3200 guarantees preservation of laminar airflow as well as easier cleaning of all cabinet surfaces. Moreover, with the majority of the system maintained outside the cabinet environment, there is no safety concern when changing optics, lasers, or PMT setup for new applications or service.

In addition, the biosafety cabinet model provides continuous The SY3200 BSC system is custom integrated with The Baker aerosol management of the sort chamber and sort collection Company SterilGARD model SG-403A HE, Class II, Type A2 area through the Baker cabinet hepa filter for a simplified Biological Safety Cabinet. The BSC is Certified Class II NSF-49 Bio- biosafety solution. The bench top system also provides an Safety standard. optional aerosol management system for the sort chamber and sort collection area.

Remote Control Configurations

The SY3200 BSC System also provides as standard the capability for remote multi-user control, especially important in BSL2 or BSL3 labs. The system can be controlled from a remote location for BSL operation with multiple workstations.

Remote control capability also enables remote service diagnostics and troubleshooting when necessary to significantly reduce downtime and Many to One service costs. Remote or BSL Operation Innovation in Laser and Light Delivery

Quality results can be directly determined by a system’s capability for both laser light management as well as emitted light collection. The SY3200 employs innovative techniques to ensure not only precise delivery of high quality laser light but also to ensure maximum emitted light collection efficiency.

Laser Delivery Array — Customizable and Scalable With its open and flexible architecture, the Laser Delivery Array (LDA) enables a broad choice of laser wavelengths and power choices.

The SY3200 offers a choice of up to 6 lasers from a selection of 15 wavelengths from leading laser manufacturers. The high quality Omicron LuxX® lasers available for the SY3200 are a small compact design that provide superior power and stability. They are available in the following wavelengths: 375, 405, 425, 445, 457, 488, 642, 685, 785nm. Additional wavelengths are available with the highly reliable Cobalt DPSS lasers: 515, 532, 561 or 594nm. The SY3200 also has an option for the versatile 552nm laser from Oxxius. A high power ultraviolet option is available from Coherent with the Genesis CX STM-Series 355nm.

This unique design allows for ideal beam spot dimensions for scatter thereby enabling optimal scatter patterns even at high data rates. The system utilizes an 809nm laser for Forward Scatter and an 830nm laser for Side Scatter.

An innovative laser delivery system provides individual beam shapers for each laser path resulting in five focused elliptical beam spots. Software controlled picomotor alignment stages for each laser allows the operator the ability to independently adjust each individual laser path without removing a single instrument cover.

Reflective Collection Optics — Superior Sensitivity The SY3200 system features the new multispot Reflective Collection Optic (RCO) design that incorporates an off axis elliptical mirror and a new revolutionary, patented multifaceted free form mirror, that provide unparalleled fluorescence collection efficiency and allow for exceptional laser spatial seperation and isolation and collimation.

The RCO ensures exceptional focusing of emitted light onto collection fiber optics for reduced crosstalk and superior sensitivity. Beams spots are spatially separated by either approximately 350 or 950μm from center spot to reduce crosstalk between lasers and different fluorescence collection channels, ultimately minimizing fluorescence compensation and background optical noise. 120 200 150 150 100 150 80 100 100 60 100 Number Number Number Number 40 50 50 50 20 0 0 0 0

-101 101 102 103 104 -101 101 102 103 104 -101 101 102 103 104 -101 101 102 103 104 BV421 FITC PE APC Measurements performed using Sphero™ Rainbow Particles RCP-30-5A Supreme Flexibility

Meeting the demand of novel dyes and increasing assay complexity requires maximum system flexibility and scalability. The detector array in the SY3200 has been designed to meet those demands with an innovative plug & play design allowing the system to accommodate any number of combinations of lasers and dyes.

Scalable PMT Array — Plug and Play Detectors The SY3200 system incorporates a customizable and Scalable PMT Array (SPA) that provides up to 30 fluorescence detectors per sorting module. In the dual module configuration, the SY3200 can have up to a total of 60 fluorescence detectors. Plug and Play PMTs and filters are easily interchangeable by the operator to allow fast setup for applications without service intervention, eliminating downtime while maximizing flexibility.

The SY3200 enables a wide range of fluorescence detection by using High Performance, red sensitive High Gain Hamamatsu HC142-02 type PMTs for all fluorescence detectors. The SPA also houses PMTs for scatter measurements for optimal scatter resolution.

Laser Wavelength

Application Dye/Fluorochrome Emmaxλ 355 375 405 445 488 515 532 561 594 642 685 SP Assay Hoechst Blue 461 455/50

Hoechst Red 670 670LP

Cell Cycle DAPI 460 455/50

Cycle / Viability PI 617 615/30

Cell Viability 7AAD 647 665/30

Cell Surface BV421 421 425/20

Pacific Blue™ 480 455/50

BV570 570 585/40

Alexa Fluor® 488 515 525/50

FITC 520 525/50

PE 575 585/40

PE-TR 615 615/30

PE-Cy5 665 665/30

PerCP 680 665/30

PerCP-Cy5.5 695 695/50

PE-Cy5.5 695 695/50

PE-Cy7 775 775/50

Alexa Fluor® 594 610 615/30

APC 660 665/30

Alexa Fluor® 647 670 665/30

Alexa Fluor® 700 720 720/40

APC-Cy7 775 775/50

FL Proteins CFP / BFP 475 455/50

EGFP 508 510/20

EYFP 527 540/30

mOrange 563 575/40

tdTomato 581 595/50

DSRed 586 595/50

mCherry 610 615/30

mKate 635 640/30

mPlum 649 650/30

Filters shown are nominal ranges. The final filter configuration may vary based on instrument setup and multicolor combinations. Powerful Electronics Processing The new Sony designed SY3200 electronics have been optimized to achieve high speed, high performance quality measurements. Sony Designed Electronics — High Resolution & Efficient High Speed Sorting

Designed by Sony, the SY3200 electronics aimed to ensure the key requirement of precise measurements at acquisition speeds of 100,000 events per second. Moreover, this new electronics design supports accurate sort decisions to be made at speeds of up to 70,000 events per second.

The electronics also enable fast pulse sampling speed of 100MHz thereby increasing precision of data measurements. Along with the high speed sampling, signal processing is accomplished by using 1x or 16x amplifiers providing a raw 32 bit data stream with an effective 24 bit resolution. As a result, the SY3200 enables up to 6 decades of data display to accommodate a wide variety of applications. This design effectively results in maximum resolution of populations throughout a large dynamic range on the histogram display.

With the ability to set multiple triggers, the electronics are compatible with unique applications requiring discrimination from more than one laser.

The electronics are capable of simultaneous acquisition of up to 32 signals, with the ability to measure the Area, Peak Height, Width, and display as Linear, Log or HyperLog transforms of each. In the dual module configuration, the electronics console can expand to accommodate up to

64 signals. 1400 2.00 1200 1.96 1000 4

10 1.95 800 3 Number 10 600 400 2 CD8-PECy7 10 200 1 10 1 0 -10 0 1000 2000 3000 4000 5000 6000 PI Area (x10) -101 101 102 103 104 CD3-BV421 CEN Double Singlet Ratio, 1.96 to 2.02 Superior Sort Performance

All aspects of the SY3200 design, high quality optics, electronics and detection combine to achieve superior sort performance. In addition, sorting is simplified through innovation and careful consideration of best sorting practices to ensure high quality, walk-away sorting.

High Speed Sorting Post sort purity of over 99% can be achieved when sorting rare populations at rates of up to 70,000 events per second.

0.88% Rare Event High Speed

0.79% 4-way Sorting 0.84% Avg. Event Rate 70K/sec 0.82%

PE Area 70μm Nozzle - 60PSI Drive Frequency – 92K

FITC Area

99.3% 99.9% 99.8% 99.7% PE Area PE Area PE Area PE Area

FITC Area FITC Area FITC Area FITC Area Flexible Sort Options Variety of sort options available including 4-way tube sorting using mixed or independent sort modes into 0.5ml microtubes, 5ml, 15ml, 50ml. Temperature control of tube sorting optionally available.

Optional plate sorting also available up to 384 well and deep well. Plate sorting uses non-deflected stream for increased accuracy and precision, especially for single cell deposition. 4 4 10 10 3 3 10 10

Sort Gate Post Sort 2 2 CD45RA-AF488 10 CD45RA-AF488 10 1 1 10 10 1 1 -10 -10

-101 101 102 103 104 -101 101 102 103 104 CD45RO-PerCP-Cy5.5 CD45RO-PerCP-Cy5.5 Enhanced Sorting Solutions Unique in its class, the SY3200 offers a choice of nozzle options that allow optimization of system performance to suit application needs.

High Performance Sensing Options The SY3200 features a Sense-in-Air configuration to provide high speed sort perfomance, that does not compromise in sensitivity and resolution.

With a choice from Ceramic tips of 70, 85, 100 and 130 μm as well as Sapphire Jewel tips ranging from 50 to 130 μm, the SY3200 can be customized for a variety of applications while maintaining superior high speed performance, high sensitivity. 4 10 4 4 10

Gate: LY 10 Post Sort 3 10 3 3 10 10 Sort Gate TCRgd-PE TCRgd-PE 2 2 10 10 CD16/56-PECy7 2 10 1 1 10 10 1 1 0 -10 -10

-101 101 102 103 104 -101 101 102 103 104 -101 101 102 103 104 CD3-BV421 CD3-BV421 CD3-BV421 High Resolution of Dim Staining NK cells High-speed High-resolution γ/δ T-cell Sorting Automated Setup & Monitoring

The SY3200 software provides automated droplet delay calculation using sortCAL beads and a unique phase lock monitoring system, monitoring satellite drop shape that allows ultimate sort control stability. iQ Monitors ensure quality sorts by automatically adjusting instrument parameters such as data rate and droplet breakoff.

If unable to maintain the sort lock, the iQ Sort Monitor will stop sample flow, cover the sort area, and warn the operator.

The SY3200 software provides 4 high quality camera displays for real-time monitoring of the instrument at either the instrument workstation or remote PC.

Besides the image of the sort stream, the operator can verify accurate sort stream trajectory as well as facilitate confirmation of amount of sample remaining. This viewing panel decreases the need for manual intervention and is ideal for integrated biosafety cabinet systems. Applications 4 Laser, 11-Color T8 Subset T4 Subset 4 4 4 6000 10 10 10 5000 3 10 3 3 10 4000 10 2 •R2: 57.82% FSC Area 3000 10 CD4-PE Area CD14-FITC Area CD14-FITC CD8-BV570 Area CD8-BV570 2000 2 1 2 10 10 1 10

•R3: 66.92% 1 1000 -10 10 1

Dump Channel Lymph Gate 1 0 -10 -10 0 102 103 104 -101 101 102 103 104 -101 101 102 103 104 -101 101 102 103 104 CD41/235--PECy5 Area CD45-AF700 Area CD3-BV421 Area CD3-BV421 Area

B Subset NK Subset HLADR NaÏve/Memory 4 4 4 4 10 10 10 10 3 3 3 3 10 10 10 10 2 2 2 CD19-APC Area CD19-APC 10 10 10 HLADR-PETR Area HLADR-PETR 2 CD16/56-PECy7 Area CD16/56-PECy7 10 CD45RA-APCCy7 Area CD45RA-APCCy7 1 0 0 0 10 1 -10

-101 101 102 103 104 -101 101 102 103 104 0 102 103 104 0 102 103 104 CD3-BV421 Area CD3-BV421 Area CD19-APC Area CD45RO-PerCPCy5.5 Area Normal whole blood was drawn into EDTA. PBMCs were isolated by centrifugation on a Ficoll gradient and stained with 12 Fluorescent Protein

monoclonal antibodies conjugated to 11 different fluorochromes. Data R2:9.83% R3:0.11% R6:12.77% R7:0.02%

was acquired on an SY3200 Cell Sorter equipped with 405, 488, 561, 4 4 10 10 and 642 nm lasers. Data analysis was performed with Winlist. Plot 1 is 3 3

ungated. Plot 2 is gated on R2. All other plots are gated on R2 & R3. 10 10 2 2 CFP 457/50 mCH 624/40 10 10 SP Assay •R2 •R6 1 1 10 10

R4:64.57% R5:25.49% R8:5.56% R9:81.65% 0 0 10 10 100 101 102 103 104 100 101 102 103 104 YFP 542/27 GFP 525/26

R2:0.97% R3:78.84% R6:91.56% R7:0.03% 4 4 10 10 3 3 10 10 2 2 CFP 457/50 mCH 624/40 10 10 •R2 •R6 1 1 10 10

R4:8.00% R5:12.19% R8:4.53% R9:3.88% 0 0 10 10 100 101 102 103 104 100 101 102 103 104 YFP 542/27 GFP 525/26 Mammalian cells (mouse hybridoma cell line), stably expressing Hoechst SP Assay with Bone Marrow Sample from a 3 week old mouse high levels of fluorescent protein genes. They were derived using a acquired on an SY3200 Cell Sorter equipped with a 375nm 20mW laser. retrovirus-mediated gene delivery system. The cells were run on an Europe SY3200 using 405nm 60mW, 488nm 80mW, 561nmnm 75mW, and 643nm 70mW lasers. Multiple Brilliant Violet Dyes Simultaneous 4 Brilliant Violet dye excitation (Brilliant Violet™ 421, Brilliant Violet™ 570, Brilliant Violet™ 605, Brilliant Violet™ 650) using a 40mW 405nm laser on an SY3200 Cell Sorter. TCRgd-PE Area TCRgd-PE CD8-BV650 Area CD14-BV570 Area CD14-BV570 CD56-BV421 Area CD20-BV605 Area CD45RO-PerCPCy5 Area CD45RO-PerCPCy5

CD3-AF647 Area CD8-BV650 Area CD3-AF647 Area CD3-AF647 Area CD3-AF647 Area CD3-AF647 Area Sony Biotechnology Inc. US / International Japan Europe

2100 South Oak Street, 5-1-12 Kita-Shinagawa, Shinagawa-ku The Heights, Brooklands, Champaign, IL 61820 Tokyo, 141-0001 Weybridge, Surrey, KT13 0XW, UK Voice: +1 800-275-5963 Voice: +0120-677010 Voice: +44 (0) 1932 816000 FAX : +1 217-328-9692 FAX : +0120-388-060 [email protected] [email protected] [email protected]

©2013 Sony Biotechnology Inc. All rights reserved. The Sony name and logo, Sony Biotechnology Inc. name and logo type, sy3200 logo and SY3200 are trademarks of Sony Corporation. Alexa Fluor® and Pacific Blue™ are registered trademarks of and licensed under patents owned by Life Technologies Corporation. Cy and CyDye are trademarks of GE Healthcare Limited. Sony Biotechnology Inc. products containing Cy5, Cy5.5 or Cy7 are manufactured under license from GE Healthcare under U.S. Patent Numbers 5,569,587; 5,627,027 and patents or pending applications that are continuations, continuations-in-part, re-examinations, divisionals, reissues or foreign equivalents thereof. Brilliant Violet, Brilliant Violet 421, Brilliant Violet 570 are trademarks of Sirigen Ltd. ClearFlow™ and ClearSort™ are trademarks of Leinco Technologies, Inc. All other trademarks are property of their respective owners. For Research Use Only. Not for use in diagnostic or therapeutic procedures.

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