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 (Canto II)

3.1. Instrument Manufacturer

BD Biosciences, San Jose, CA (

3.2. Instrument Model

BD Custom FACS Canto II Flow Cytometer, Serial number V96300457

Technical specifications at:

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:

LASERFLUOROCHROMEPMTLP MIRRORBP FILTER VOLTAGE

FSCdiodenonenone

488 nmSSCFnone488/10

FITCE502530/30

PE D556585/42

NoneC610nonen/a

PerCP-Cy5.5B655670LP

PE-Cy7A735780/60

633 nmAPCCnone660/20

NoneB685nonen/a

APC-Cy7/H7A735 780/60

407 nmPacific BlueBnone450/50

Pacific OrangeA 502none

3.4. Other Relevant Instrument Details

BD FACS Canto II User’s Guide:

BD FACSDiva Software Reference Manual:
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)