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 / TiterCD45 / Anti-CD45 / FITC / BD Biosciences / 347463 / 10001 / 5 mL
3. Instrument Details (Canto II)
3.1. Instrument Manufacturer
BD Biosciences, San Jose, CA (http://www.bdbiosciences.com/home)
3.2. Instrument Model
BD Custom FACS Canto II Flow Cytometer, Serial number V96300478
Technical specifications at: http://www.bdbiosciences.com/pdfs/whitePapers/23-8786-01.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; three-laser configuration as follows:
· 488-nm Coherent® SapphireTM air-cooled argon-ion laser; 20 mW
· 633-nm JDS UniphaseTM HeNe air-cooled laser; 17 mW
· 405-nm Coherent® solid state diode laser; 30 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 a BD octagon (488 nm laser line) and BD Trigons (633 and 405 nm laser lines). PMTs are arranged as follows:
LASER FLUOROCHROME PMT LP MIRROR BP FILTER VOLTAGE
FSC diode none none
488 nm SSC F none 488/10
FITC E 502 530/30
PE D 556 585/42
None C 610 none n/a
PerCP-Cy5.5 B 655 670LP
PE-Cy7 A 735 780/60
633 nm APC C none 660/20
None B 685 none n/a
APC-Cy7/H7 A 735 780/60
407 nm Pacific Blue B none 450/50
Pacific Orange A 502 510/50
3.4. Other Relevant Instrument Details
BD FACS Canto II User’s Guide: http://hsc.usf.edu/NR/rdonlyres/E9C6F213-BF6C-4AA4-9A72-0B2612BA1412/27228/CantoManual.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)