Multicolor Flow Cytometry for HIV-p24 Analysis

Surface staining for flow cytometry analysis was performed with the following fluorochrome-conjugated antibodies (Abs): CD3 Pacific blue (clone UCHT1), CD4 Alexa700 (clone RPA-T4; BD Biosciences), and CD45RA APC-eFluor 780 (clone HI100; eBioscience). Intracellular staining was performed with FITC-conjugated HIV-p24 Abs (clone KC57; Beckman Coulter) and the Fixation/Permeabilization Kit (BD). The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) was used to exclude dead cells. Flow-cytometry analysis was performed using a LSRII cytometer, Diva version 8 (BD Biosciences, San Jose, CA, United States), and FlowJo version 10.0.6 (Tree Star, Inc.). Flow cytometry gates were defined using the fluorescence minus one (FMO) strategy (Roederer, 2002 (link); Gosselin et al., 2010 (link)).

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Zhang Y., Planas D., Raymond Marchand L., Massanella M., Chen H., Wacleche V.S., Gosselin A., Goulet J.P., Filion M., Routy J.P., Chomont N, & Ancuta P. (2020). Improving HIV Outgrowth by Optimizing Cell-Culture Conditions and Supplementing With all-trans Retinoic Acid. Frontiers in Microbiology, 11, 902.

Publication 2020

CD3 Pacific blue (clone UCHT1), CD45RA APC-eFluor 780 (clone HI100; Fixation/Permeabilization Kit LIVE/DEAD Fixable Aqua Dead Cell Stain Kit LSRII cytometer Diva version 8

Antibodies Antibodies hiv Cells Clone Fitc Flow cytometry Fluorescence Fluorochrome Hiv p24 Intracellular Stain Tree

Corresponding Organization : Université de Montréal

Other organizations : Caprion (Canada), Alethia Biotherapeutics (Canada), McGill University Health Centre

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Variable analysis

independent variables

Type of fluorochrome-conjugated antibodies used for surface staining: CD3 Pacific blue, CD4 Alexa700, CD45RA APC-eFluor 780
Type of FITC-conjugated antibody used for intracellular staining: HIV-p24

dependent variables

Flow cytometry analysis of surface and intracellular markers on cells

control variables

Use of LIVE/DEAD Fixable Aqua Dead Cell Stain Kit to exclude dead cells
Use of the Fixation/Permeabilization Kit for intracellular staining
Flow cytometry analysis performed using a LSRII cytometer, Diva version 8, and FlowJo version 10.0.6
Flow cytometry gates defined using the fluorescence minus one (FMO) strategy

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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