Anti cd4 percp clone l200

Flow cytometry was performed as described previously (6 (link)). Isolated cells were incubated with predetermined optimized concentrations of anti-CD3-Alexa fluor 700 (clone SP34-2, BD Pharmingen), anti-CD8-Pacific Blue or PE-CF594 (clone RPA-T8, BD Pharmingen), anti-CD20-APC-Cy7 (clone L27, BD Pharmingen), anti-CXCR5-phycoerythrin (PE) (Clone MU5UBEE, eBioscience), anti-CD200-APC (clone OX104, eBioscience), anti-CD4-PerCP (clone L200, BD Pharmingen), anti-CD95-PE-Cy5 (clone DX2, BD Pharmingen), anti-CCR7-APC-Cy7 (clone 3D12, Biolegend), anti-PD-1-PE-Cy7 (clone EH12.2H7, Biolegend), anti-ICOS-Pacific Blue (C398.4A, Biolegend) and Live/Dead dye-Alexa430 (Invitrogen). After staining the cell surface, cells were permeabilized and fixed by BD perm wash (BD Pharmingen) and washed twice. Finally, cells were incubated with anti-Ki67-FITC (B56, BD Pharmingen), washed twice and diluted in 1% PFA. Data were acquired on a LSRII flow cytometer (BD Biosciences) and the data analyzed using FlowJo software (version 9.2 Tree Star).

PBMC were stained with CFSE (CellTrace^™ CFSE Cell Proliferation Kit, Invitrogen) and incubated with or without SIVmac239 Gag peptides (2 μg/ml for each peptide). The peptides (obtained through ARRRP) were 15-mers with an 11-amino acid overlap between sequential peptides and represented the complete protein sequence. Cells without any stimuli were used to determine background proliferation. After incubation for 5 days at 37 °C, cells were stained with anti-CD3-Alexa Fluor 700 (clone SP34-2), anti-CD4-PerCP (clone L200), and anti-CD8-PE (clone RPA-T8) Abs (all from BD Pharmingen). After fixation, at least 10,000 CD3⁺ cells were acquired by flow cytometry, and data were analyzed using FACS-Diva (BD Biosciences) software. The percentages of proliferating CD3⁺CD4⁺ and CD3⁺CD8⁺ cells were determined by CFSE dilution; background proliferation (without stimulation) was subtracted.