Cd45ro pacific blue

For suppression assays, cells were washed with 0.1% (w/v) sodium azide/phosphate-buffered saline (Mediatech Cellgro) on day 7 of in vitro stimulation and were stained with anti-CD4 PECy5 (BD Pharmingen), anti-CD8 Pacific Blue (Biolegend), and anti-CD25 APCCy7 (BD Pharmingen. For surface phenotyping of cells, bulk PBMCs and enriched CD8+ T cells were stained with anti-CD3 Alexa 700 (BD Pharmingen), anti-CD8 AmCyan (BD Biosciences), anti-CD27 APCCy7 (Biolegend), anti-CD28 APC (BD Pharmingen), CD45RO Pacific Blue (Biolegend), anti-CD62L PECy5 (BD Pharmingen), and anti-CD57 PE (Southern Biotech). For intracellular staining of cytokines, cells were initially activated with 1 μL of leukocyte activation cocktail (BD Pharmingen) for 5 hours. Cells were surface stained with anti-CD8 APC (BD Biosciences), anti-CD4 PECy5 (BD Pharmingen) and anti-CD25 APCCy7 (BD Pharmingen) and permeabilized as described previously. Intracellular staining was performed using anti-IFNγ PECy7 (BD Pharmingen), anti-IL-17A PE (Ebioscience), anti-Granzyme B Alexa 700 (BD Pharmingen) and anti-Perforin Pacific Blue (BD Pharmingen). All cells were resuspended in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfiled, PA) for FACS analysis. Flow cytometric data were acquired on a 4-Laser, 17-color LSRII using FACSDiva software (Becton Dickinson). CFSE was detected in the FITC channel on the LSR.