1. Experiment Overview

1. Experiment Overview 1.1. Purpose

1.2. Keywords

1.3. Experiment Variables

1.4. Organization 1.4.1. Name: Baylor Institute for Immunology Research 1.4.2. Address: 3434 Live Oak Street, Dallas, TX 75204 1.5. Primary Contact 1.5.1. Name: 1.5.2. Email Address : 1.6. Date

1.8. Quality Control Measures 2. Flow Sample/Specimen Details 2.1. Sample/Specimen Material Description 2.1.1.1. Biological Sample Description: 2.1.1.2. Biological Sample Source Description: 2.1.1.3. Biological Sample Source Organism Description: 2.1.1.3.1. Taxonomy: 2.1.1.3.4. Phenotype: 2.1.1.3.5. Genotype: 2.1.1.3.6. Treatment: 2.3. Sample Treatment(s) Description

2.4. Fluorescence Reagent(s) Description

Analyte Detector Fluorochrome Manufacturer Catalog Number Lot Number Titer CD45 Anti-CD45 FITC BD Biosciences 347463 10001 5 L 3. Instrument Details (LSRII) 3.1. Instrument Manufacturer BD Biosciences, San Jose, CA (http://www.bdbiosciences.com/home) 3.2. Instrument Model BD Special Order LSRII Flow Cytometer, Serial number H47100060 Technical specifications at: http://www.bdbiosciences.com/pdfs/whitePapers/23-8641-00.pdf 3.3. Instrument Configuration and Settings 3.3.1. Flow Cell and Fluidics: The instrument has not been altered; fixed-alignment cuvette flow cell. 3.3.2. Light Sources: The instrument has not been altered; four-laser configuration as follows:  488-nm Coherent® SapphireTM air-cooled argon-ion laser; 20 mW  532-nm Coherent® M315 air-cooled laser; 150 mW variable power  638-nm Coherent® Cube air-cooled laser; 30 mW  405-nm Coherent® Radius air-cooled laser; 50 mW 3.3.3. Excitation Optics Configuration: The instrument has not been altered. 3.3.4. Optical Filters: See figure below. 3.3.5. Optical Detectors: The instrument has not been altered. See detector voltages below. 3.3.6. Optical Paths: The instrument has not been altered. Detector arrays consist of BD octagons (532 and 405 nm laser lines) and BD Trigons (488 and 638 nm laser lines). PMTs are arranged as follows:

LASER FLUOROCHROME PMT LP MIRROR BP FILTER VOLTAGE FSC diode none none 488 nm SSC C none 488/10 FITC B 505 525/50 PerCP-Cy5.5 A 670 695/40 532 nm PE E none 575/26 PE-Texas Red D 600 610/20 PE-Cy5 C 635 660//20 PE-Cy5.5 B 685 710/50 PE-Cy7 A 735 780/60 638 nm APC C none 660/20 AlexaFluor 700 B 685 710/50 APC-Cy7/H7 A 755 780/60 405 nm Pacific Blue H none 450/50 Am Cyan G 505 525/50 Pacific Orange F 535 560/40 Qdot 585 E 570 585/42 Qdot 605 D 600 605/40 Qdot 655 C 630 660/40 Qdot 705 B 670 710/50 Qdot 800 A 735 780/60

3.4. Other Relevant Instrument Details BD LSRII User’s Guide: http://facs.stanford.edu/sff/doc/BDLSRIIUserGuide.pdf BD FACSDiva Software Reference Manual: http://facs.stanford.edu/sff/doc/bdfacsdivasoftwareuserguide.pdf 4. Data Analysis Details 4.1. List-mode Data Files FCS files can be obtained by contacting: 4.2. Compensation Details Compensation has been performed post-acquisition according to the following spillover matrix: (paste matrix from FlowJo here) 4.3. Data Transformation Details 4.3.1. Purpose of Data Transformation: visualization and gating 4.3.2. Data Transformation Description: FlowJo default visualization settings have been used for gating: Parameters to display with logarithmic scaling: All fluorescence parameters Linear channel number corresponding to left edge of log display: 3 Number of decades to display log-converted data: 5 for pulse area parameters, 4 for pulse height parameters Logicle transformation enabled: number of decades: 4.5, additional negative display size: 0, width basis: -10. 4.3.3. Other relevant data transformation details: FlowJo v. 8.4.5 for Mac OSX was used. 4.4. Gating (Data Filtering) Details 4.4.1. Gate Description

4.4.2. Gate Statistics

4.4.3. Gate Boundaries (or images or detailed gate membership information) (attach FlowJo workspace files here)