Polychromatic Flow Cytometry of Immune Markers

The following fluorochrome-conjugated antibodies (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, California, USA), CCR7-FITC (150503) (R&D Systems, Minneapolis, Minnesota, USA), CD45RA-APC-eFluor780 (HI100) (eBioscience, San Diego, California, USA), CD3-Alexa700 (UCHT1), CD326-BV650 (9C4), CD8-PerCP-Cy5.5 (RPA-T8), CD19-PerCP-Cy5.5 (HIB19), CD66b-PerCP-Cy5.5 (G10F5), human leukocyte antigen - antigen D related (HLA-DR)-BV785 (L243; Biolegend ,San Diego, California USA), CD4-FITC (SFCI12T4D11), and HIV-p24-RD1 (FH190–1–1); (Beckman Coulter, Fullerton, California, USA). Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid; Life Technologies, Burlington, Ontario, Canada) was used to exclude dead cells. Cells were analyzed by fluorescence-activated cell sorting (FACS) using the BD-LSRII cytometer and BD-Diva (BD Biosciences) and FlowJo (Tree Star, Inc., Ashland, Oregon, USA) softwares. All Abs were titrated for an optimal noise/signal ratio and Ab cocktails were validated by comparing single with multiple staining. Positive gates were placed based on fluorescence minus one 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 CCR7-FITC (150503) CD45RA-APC-eFluor780 (HI100) CD3-Alexa700 CD326-BV650 CD8-PerCP-Cy5.5 CD19-PerCP-Cy5.5 CD66b-PerCP-Cy5.5 (G10F5), HLA-DR)-BV785 CD4-FITC (SFCI12T4D11), HIV-p24-RD1 (FH190–1–1); BD-LSRII cytometer

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

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Variable analysis

independent variables

Antibodies (Abs) used for polychromatic flow cytometry analysis

dependent variables

Cellular phenotypes and expression of surface markers (CD3, CD4, CCR4, CCR6, CCR7, CXCR3, CD45RA, CD326, CD8, CD19, CD66b, HLA-DR, HIV-p24)

control variables

Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid)
Fluorescence minus one controls for positive gate placement

controls

Positive controls: Fluorescence minus one controls
Negative controls: Not explicitly mentioned

Annotations

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