Markers of T-cell activation (HLA-DR, CD69, and CD25) that have been associated with HIV latency [49 (link)–53 (link)], were measured using flow cytometry. Briefly, freshly isolated PBMCs were stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Invitrogen) and then stained with the following fluorescently-conjugated monoclonal antibodies: CD3 BV421 (clone UCHT1), CD4 PE-Cy7 (clone RPA-T4), CD25 BV605 (clone BC96), HLA-DR FITC (clone L243) from Biolegend. CD69 PE (clone FN50) from BD Biosciences. Stained cells were fixed in PBS with 1% paraformaldehyde, and stored in the dark at 4°C until acquisition. All phenotyping data were collected on BD LSR II (BD Biosciences). Data were analyzed using FlowJo software (Treestar).
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Quantifying T-cell Activation Markers in HIV Latency
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Corresponding Organization : The Wistar Institute
Other organizations : Science Research Laboratory, Genos (Croatia), University of Pennsylvania, Philadelphia Fight, Rush University Medical Center, Albert Einstein College of Medicine, University of Zagreb
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Variable analysis
independent variables
- HLA-DR
- Markers of T-cell activation
- Freshly isolated PBMCs
- LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Invitrogen)
- CD3 BV421 (clone UCHT1)
- CD4 PE-Cy7 (clone RPA-T4)
- CD25 BV605 (clone BC96)
- HLA-DR FITC (clone L243)
- CD69 PE (clone FN50)
- PBS with 1% paraformaldehyde
- BD LSR II (BD Biosciences)
- FlowJo software (Treestar)
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