HIV Infection Analysis by Flow Cytometry

Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD8-FITC, CD4-FITC, CD4-PE, CD3-APC, CD3-APC-Cy7 (eBioscience, San Diego, CA), CCR5-PE-Cy7 (BD Biosciences, San Jose, CA), CCR5-PE (R&D Systems, Minneapolis, MN), CCR6-PerCp-Cy5.5, CD90 Alexa Fluor-647 (Biolegend, San Diego, CA). For the HIV-infection experiments, following infection for 6–7 days, intracellular levels of p24 were analyzed as described previously^{8 (link)}. Briefly, after surface staining, cells were washed, fixed and permeabilized (20 min) following instructions provided in the Cytofix/Cytoperm Plus kit (BD Biosciences) and stained for intracellular p24 with KC57-FITC antibody (Beckman Coulter; Danvers, MA) for 30 min. Analysis was performed on BD FACSCalibur or BD FACSCanto flow cytometers (BD Biosciences) using FACSdiva software, and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers was measured by the percentage of positive cells and the mean fluorescence intensity (MFI).

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Rodriguez-Garcia M., Barr F.D., Crist S.G., Fahey J.V, & Wira C.R. (2014). Phenotype and Susceptibility to HIV-infection of CD4+ Th17 Cells in the Human Female Reproductive Tract. Mucosal immunology, 7(6), 1375-1385.

Publication 2014

CD8-FITC CD4-FITC CD4-PE CD3-APC CD3-APC-Cy7 CCR5-PE-Cy7 CCR5-PE CCR6-PerCp-Cy5.5 CD90 Alexa Fluor-647 Cytofix/Cytoperm Plus kit KC57-FITC antibody BD FACSCalibur BD FACSCanto

Alexa fluor 647 Antibodies Antibody Ccr5 Ccr6 Cd90 Cells Cy5 5 Fitc Fluorescence Hiv infection Infection Intracellular Tree

Corresponding Organization :

Other organizations : Dartmouth College

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Variable analysis

independent variables

HIV infection

dependent variables

Intracellular levels of p24
Expression of surface markers (percentage of positive cells and mean fluorescence intensity (MFI))

control variables

Mixed cell suspensions
Combinations of the following antibodies: CD8-FITC, CD4-FITC, CD4-PE, CD3-APC, CD3-APC-Cy7, CCR5-PE-Cy7, CCR5-PE, CCR6-PerCp-Cy5.5, CD90 Alexa Fluor-647
Cytofix/Cytoperm Plus kit for fixation and permeabilization
KC57-FITC antibody for intracellular p24 staining
Flow cytometry analysis on BD FACSCalibur or BD FACSCanto flow cytometers using FACSdiva software, and data analyzed with FlowJo software

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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