Allophycocyanin cy7 anti cd8

Flow cytometry was used to detect CD3⁺, CD4⁺ and CD8⁺ cells. PBMCs were collected, washed for 3 min using PBS at room temperature and centrifuged at 200 × g for 5 min at room temperature and the supernatant was discarded. FITC-anti-CD3 (1:200; cat. no. 561806; BD Biosciences), phycoerythrin-anti-CD4 (1:200; cat. no. 561843; BD Biosciences) and allophycocyanin-Cy7-anti-CD8 (1:200; cat. no. 557760; BD Biosciences) antibodies were added, followed by incubation at 4°C for 20 min. PBMCs were washed for 5 min using PBS at room temperature. Following centrifugation at 200 × g for 5 min at room temperature, the PBMCs were resuspended with PBS containing 1% paraformaldehyde for fixation at room temperature, detected (FACSCanto II; BD Biosciences) and analyzed by CellQuest software v.5.1 (BD Biosciences).