Cd8a percp

Cells (1–2 × 10⁶) were dispensed into a 96-well u-bottom plate and washed in staining buffer (PBS + 1% bovine serum albumin +0.1% sodium azide). Cells were re-suspended in staining buffer containing anti-rat CD32 antibody (clone D34–485) for blocking of Fc receptor (BD Biosciences; San Jose, CA). Cells were next resuspended in staining buffer containing a cocktail of biotin and fluorochrome-conjugated mouse anti-rat antibodies specific for cell surface antigens: CD3-FITC (clone G4.18), CD4-APC (OX-35), CD8a-PerCP (OX-8), CD11b-V450 (WT.5), CD45RA-PE (OX-33) and CD161a-Biotin (10/78)(BD Biosciences). Cells were washed with staining buffer and re-suspended in staining buffer containing streptavidin APC-Cy7 secondary (BD Biosciences). Following an incubation, cells were washed twice in staining buffer and fixed in Cytofix buffer according to the manufacturer’s instructions (BD Biosciences). Within 24 h, cells were re-suspended in staining buffer and analyzed on an LSR II flow cytometer (BD Biosciences). Data analysis was performed with FlowJo 7.6.5 software (TreeStar Inc.; Fenton, MI). Cells gated on single cells using SSC-A × SSC-H doublet discrimination were identified as CD4 T cells (CD4+ CD3+), CD8 T cells (CD8+ CD3+), B cells (CD45RA+ CD3-), NK cells (CD161a^hi CD3-), and CD11b + myeloid cells.