Cd3 pe cy7 ucht1

Red blood cells from whole blood were lysed with 4.5 ml of water for 15 s before adding 0.5 ml of 10X PBS. Red blood cells from spleen or bone marrow were lysed with ACK buffer (NH 4 Cl 0.15 M, KHCO3 10 mM, EDTA 0.1 mM). Cell suspensions were stained with an optimal quantity of antibodies at a concentration of 10 7 cells/mL in a final volume of 100 µL of PBS/FCS 3%. Incubation was performed in the dark at 6°C for 20 min. The following anti-human mAbs were used for cell surface staining: CD45 PE-CF594 (clone HI30; catalog number (cat ≠) 562279, BD Biosciences) anti-CCR5 Alexa Fluor 647 (HEK/1/85a; cat ≠ 313712, Biolegend), anti-CD4 PerCP (RPA-T4, cat ≠ 300528, Biolegend), anti-CD8 Alexa Fluor 700 (HIT8a, cat ≠ 300920, Biolegend), CD3 PE-Cy7 (UCHT1, cat≠ 300420, Biolegend). The human IgG1 mAb 2F5 specific for a gp41 epitope (cat ≠ AB001, Polymun, Austria) was used to detect the C46 peptide. The KC57-RD1 (cat≠ 6604667, Beckman Coulter) antibody was used to detect intracellular p24 after cells were treated with permeabilization buffer (eBioscience, fixation and permeabilization kit). All cell preparations were acquired on an LSRII cytometer (BD) and analyzed with FlowJo software (Tree Star, Portland, OR). The frequencies of positive cells were determined according to the fluorescence minus one (FMO) staining negative control.