PBMC were thawed and batch analyzed using the Becton Dickson Facs Canto II flow cytometer (BD Biosciences) at the Infectious Diseases Institute and housed in the Immunology laboratory at Makerere University College of Health Sciences. Cell surface and intracellular staining were performed to measure expression of markers of immune activation and regulatory T-cells using antibodies CD3, CD4, CD38, HLADR, CD25 and FoxP3 (BD Biosciences) and samples were acquired using a Facs Canto II flow cytometer. In general, at least 300,000 events were collected. Gating was standardized and set using fluorescence minus one controls (FMOs) for HLADR, CD38, CD25 and FoxP3 isotype (Figure 1 ). Regulatory T-cells were defined as CD3+CD4+FoxP3+CD25+bright cells, and immune activation was defined as CD3+CD4+CD38+ HLA DR+ cells.
Becton dickson facs canto 2 flow cytometer
Manufactured by BD
Sourced in United States
The Becton Dickson Facs Canto II is a flow cytometer. It is a laboratory instrument used for the analysis of particles, including cells, in a fluid as they pass through a beam of light.
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2 protocols using becton dickson facs canto 2 flow cytometer
1
Flow Cytometric Analysis of PBMC
2
Multiparametric Flow Cytometry of Immune Cells
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Single cell suspensions of isolated brain leukocytes, splenocytes, or leukocytes were resuspended, and stained with fluorochrome-conjugated antibodies. Cells were evaluated by surface staining of the with Pacific Blue™ rat anti-mouse CD3e, APC-Cy™7 rat anti-mouse CD19, PE-Cy™7 rat anti-mouse CD8, APC rat anti-mouse MHC Class II (I-A/I-E), PerCP/Cy5.5 rat anti-mouse CD4, and FITC mouse anti-mouse CLIP (15G4) along with LIVE/DEAD® Fixable Aqua Dead Cell Stain. The cells were analyzed on a Becton Dickson FACSCanto II flow cytometer (BD Biosciences Inc., San Jose, CA, USA), consisting of a 3 laser 10 parameter system with FACSDiva software (BD Biosciences Inc., San Jose, CA, USA). The flow data was analyzed using FlowJo® software (FlowJo, LLC, Ashland, OR, USA). For all flow cytometry, samples are coded prior to running through the FACS cell sorter. Once gating strategies have been applied consistently across all groups and data have been collected, the codes are broken for statistical analysis.
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