Polychromatic Flow Cytometry Analysis

The following fluorochrome-conjugated Abs were used for polychromatic flow cytometry analysis: CD3-Pacific-blue (UCHT1), CD4-Alexa-Fluor 700 (RPA-T4), CCR4-PE-Cy7 (1G1), CCR6-PE (11A9), CCR7-PE-Cy7 (3D12), CXCR3-PE-Cy5 (1C6) (BD Biosciences, San Diego, CA), CCR7-FITC (150503) (R&D Systems, Minneapolis, MN), CD45RA-APC-eFluor780 (HI100) (eBioscience, San Diego, CA), CD3-Alexa700 (UCHT1), CD326-BV650 (9C4), CD8-PerCP-Cy5.5 (RPA-T8), CD19-PerCP-Cy5.5 (HIB19), CD66b-PerCP-Cy5.5 (G10F5), HLADR-BV785 (L243; BioLegend), CD4-FITC (SFCI12T4D11) and HIV-p24-RD1 (FH190-1-1; Beckman Coulter, Fullerton, CA). Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid; Life Technologies, Burlington, ON) was used to exclude dead cells. Cells were analyzed by FACS using the BD-LSRII cytometer and BD-Diva (BD Biosciences, San Jose, CA), and FlowJo (^©Tree Star, Inc., Ashland, OR) softwares. All Abs were titrated for an optimal noise/signal ratio and Ab cocktails were validated by comparing single to multiple staining. Positive gates were placed based on fluorescence minus one (FMO) controls [41 (link), 69 ]

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Gosselin A., Wiche Salinas T.R., Planas D., Wacleche V.S., Zhang Y., Fromentin R., Chomont N., Cohen É.A., Shacklett B., Mehraj V., Ghali M.P., Routy J.P, & Ancuta P. (2016). HIV persists in CCR6+CD4+ T cells from colon and blood during antiretroviral therapy. AIDS (London, England), 31(1), 35-48.

Publication 2016

CD3-Pacific-blue CD4-Alexa-Fluor 700 CCR4-PE-Cy7 (1G1), CCR7-FITC (150503) CD45RA-APC-eFluor780 (HI100) CD3-Alexa700 CD8-PerCP-Cy5.5 CD19-PerCP-Cy5.5 CD66b-PerCP-Cy5.5 (G10F5), HLADR-BV785 CD4-FITC HIV-p24-RD1 (FH190-1-1 BD-LSRII cytometer

Ccr6 Cd326 Cd66b Cells Cxcr3 Cy5 5 Fitc Flow cytometry Fluorescence Fluorochrome Hiv p24 Stain Tree

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Variable analysis

independent variables

Type of fluorochrome-conjugated Abs used for polychromatic flow cytometry analysis

dependent variables

Measurements obtained from polychromatic flow cytometry analysis

control variables

Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid) used to exclude dead cells
Antibody cocktails validated by comparing single to multiple staining
Positive gates placed based on fluorescence minus one (FMO) controls

controls

Fluorescence minus one (FMO) controls used to place positive gates

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