Cd3 pacific blue clone ucht1

Freshly isolated BALF cells were stained ex vivo using the following antibodies: CD3-Pacific Blue, clone UCHT1 (BD Pharmingen, San Diego, CA, USA), CD4-APC-H7, clone SK3 (BD Biosciences, San Jose, CA, USA), Vα2.3-FITC, clone F1 (Thermo Scientific, Rockford, IL, USA), and Vβ22-PE, clone IMMU 546 (Beckman Coulter Immunotech, Marseille, France). Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies, Eugene, OR, USA) was used for assessment of cell viability. Cells were sequentially gated on lymphocytes (based on FSC vs. SSC), single cells (based on FSC-A vs. FSC-H), viable cells (defined as Aqua negatively stained cells), CD3⁺, and CD4⁺ cells. The CD4⁺ gate was set as threshold gate for acquisition, with a minimum of 15,000 events being collected. Flow cytometry was run on a BD FACSVerse (Beckton Dickinson, San Jose, CA, USA) and results were analyzed using FlowJo X (TreeStar, Ashland, OR, USA) software.

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)).

The following anti-human antibodies were used for staining of cytobrush and whole blood samples: CD3-Pacific Blue (clone UCHT1, BD), CD4-PerCP Cy5.5 (clone OKT4, eBioscience), CD8-Horizon-V500 (clone RPA-T, BD), CD45-APC-H7 (clone 2D1, BD), CD45RO-PE (clone UCHL1, BD), HLA-DR-APC (cloneG46-6 BD-Pharmingen) (only for cytobrush samples), α4β7-FITC (clone FIB504, Biolegend), and CCR5-PEcy7 (clone 2D7/CCR5, BD). Cells were incubated with the antibody cocktail at 4°C for 30 minutes in the dark, washed with FACS wash buffer (PBS with 1% bovine serum albumin and 0.05% sodium azide), and acquired on a FACS Canto II (BD) after fixation with 2% paraformaldehyde in water. Only cervical cytobrush samples without visible red blood cells, satisfactory staining quality, and T-cell counts above 150 cells were included in the analysis. For whole blood, a cut-off of at least 10,000 lymphocyte events was applied. Fluorescent spill over compensation was conducted with antibody capture beads (BD) stained separately with the individual antibodies used in the test samples. Gating was guided for both sample types (peripheral blood and cytobrush) separately by fluorescence minus one (FMO) controls. Flow cytometry data was processed using FlowJo version 10.4 (Tree Star Inc.).