Mouse anti chicken cd8 rpe

Peripheral blood was taken from wing veins with heparin and washed twice with cold PBS by centrifugation at 300 g at 4°C for 5 min and resuspension in cold PBS. Cells were counted using a hemocytometer, and around 5 × 10⁷ lymphocytic cells in 2 ml were stained at 4°C in the dark for 1 h using T cell specific antibodies [10 µl mouse anti-chicken CD4-FITC and 10 µl of mouse anti-chicken CD8b-FITC for lines N and P2a; 10 µl mouse anti-chicken CD4-RPE and 10 µl of mouse anti-chicken CD8-RPE for lines 15I and 6₁ (all antibodies from Southern Biotech)]. Then the cells were washed 3–4 times with cold PBS and resuspended into 1 ml cold PBS for sorting, using magnetic-activated cell sorting (MACS, Miltenyi Biotec) for line N and P2a, and a DakoCytomation MoFlo MLS high-speed cell sorter (Beckman Coulter) for fluorescence-activated cell sorting (FACS, performed by Mr. Nigel Miller in the Department of Pathology) for line 15I and 6₁.