Pe cy5 conjugated anti cd8

Cryopreserved PBMCs were thawed at 37°C, washed twice with PBS, and stained with trypan blue to determine cell viability. PBMCs were adjusted to a concentration of 1×10⁶/ml in RPMI1640 medium supplemented with 10% fetal bovine serum. The PBMCs were incubated for 4 hours at 37°C, 5% CO₂ in the presence of PMA/Ionomycin mixture (Liankebio, LK-CS1001) and BFA/Monensin mixture (Liankebio, LK-CS1002) according to the manufacturer’s instructions. After incubation, the cells were stained with FITC-conjugated anti-CD3 (BD, USA) and PE-Cy5-conjugated anti-CD8 (Biolegend, 301010) to delimitate CD4⁺ T cells. Then the cells were stained with PE-conjugated anti-IL-17A (Biolegend, 512306) for Th17 detection after fixation and permeabilization (BD IntraSure Kit, 4043524). Stained cells were tested on a FACS Calibur flow cytometer (BD, USA) and then analyzed using CellQuest software (BD Bioscience).

FITC-conjugated anti-CD3, APC-conjugated anti-CD4, APC-conjugated anti-CD8, and their corresponding isotype controls were purchased from eBioscience (San Diego, CA). PE-Cy5-conjugated anti-CD4, PE-Cy5-conjugated anti-CD8, FITC-conjugated anti-CD44, and their corresponding isotype controls, were obtained from BioLegend (San Diego, CA). PE-conjugated anti-CD45RB, PE-conjugated anti-Fas, and their corresponding isotype controls were purchased from BD Bioscience (San Jose, CA). FITC-conjugated anti-CD28 and its corresponding isotype control were purchased from Serotec (Oxfordshire, UK). Rabbit polyclonal primary anti-Ki-67 and its isotype control were purchased from Abcam (Cambridge, UK) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. PerCP-conjugated donkey anti-rabbit IgG antibody was obtained from Santa Cruz Biotechnology. T cell mitogen concanavalin A (Con A) was obtained from Sigma (St. Louis, MO).