Cd4 pe cy7

Whole blood was collected in 10 mL purple top vacutainer tubes containing EDTA (BD Biosciences, Franklin Lakes, NJ, USA). PBMCs were isolated from whole blood by density gradient centrifugation. The blood was layered on top of 4 mL of Ficoll-Paque™ Plus (GE Healthcare, Piscataway, NJ, USA) in 15 mL tubes and centrifuged for 30 min at 400g with the brake set to off. PBMCs were collected, washed twice with sterile phosphate-buffered saline (PBS), resuspended in freezing medium (fetal bovine serum (GIBCO) with 10% dimethylsulfoxide (DMSO), and store at − 80 °C until further use.
For cell surface staining, PBMCs were thawed and washed three times with PBS. Cells were counted and distributed equally (4 ×10⁵ cells / FACS tubes). Cells were then stained with CD66e-FITC (Abcam), CD26-PE (BD biosciences, USA), CD4-PE-Cy7 (Abcam), CD8-APC (Abcam), CD14-PerCp (Meltini biotec, Germany) antibodies and incubated in the dark for 20 min at 25 °C. The cells were then washed once and run using the BD Biosciences LSRII flow cytometry (BD Biosciences, USA). Data were analyzed using FACS diva software (BD Biosciences, USA). For CD66e and CD26 expression on monocytes, the gating was performed based on CD14 positive within monocytes gate. For CD66e and CD26 expression on lymphocytes, the gating was performed based on CD4 positive or CD8 positive within lymphocytes gate.