Hcd4 percp cy5

Vaginal, splenic, and PBMCs were isolated as above and stained for 30 minutes with a cocktail of antibodies [hCD45 – V450, hCD4 – PerCP-Cy5.5, hCD8 – PE-Cy7 (BD Biosciences, San Jose, CA, USA); hCD3 – BV605 (Biolegend, San Diego, CA, USA); mCD45 – AF700 (eBioscience, San Diego, CA, USA)], following 20 minutes of Fc receptor blocking (eBioscience, San Diego, CA, USA) on ice. For intracellular staining (p24-antigen), cells were subsequently permeabilized and fixed with BD Cytofix/Cytoperm^TM Solution (BD Biosciences, San Jose, CA, USA) for 20 minutes, followed by a 30 minute incubation with the intracellular p24 antibody [p24 – PE (Beckman Coulter, Mississauga, ON, Canada)]. Within 24 hours of staining, data was acquired on the BD LSRII or BD LSR Fortessa flow cytometer (BD Biosciences, Canada). Data was analyzed and cell frequencies and phenotype were assessed using FlowJo® software (Version 10.0.8) (Treestar, Ashland, OR, USA).

Blood was collected by cheek bleed (as above) or at experimental endpoint by cardiac puncture as previously described^{40 (link)}. PBMCs were recovered from blood following ACK lysis buffer (2 mL, 4 min; then 500 μL 1 min), and neutralized with PBS. The resulting cell preparations were counted on a hemocytometer, resuspended to a final concentration of 1–3 × 10⁶ cells/mL, and stained for flow cytometry.
PBMCs were stained for 30 min with antibodies [hCD45—V450, hCD4—PerCP-Cy5.5, hCD8—PE-Cy7 (BD Biosciences, San Jose, CA, USA); hCD3—BV605, hCCR5—AF647 (Biolegend, San Diego, CA, USA); mCD45—AF700 (eBioscience, San Diego, CA, USA)], following 20 min of Fc receptor blocking (eBioscience, San Diego, CA, USA) on ice. Data were immediately acquired on a BD LSRII flow cytometer (BD Biosciences, Canada) and results were analyzed using FlowJo software (Version 10.0.8) (Treestar, Ashland, OR, USA). Gating strategy is presented in Supplemental Fig. 2.