Cd8 percp cy5.5 rpa t8

Whole fresh blood samples were prepared immediately after collection for immunophenotyping. Immune cell populations were characterized by surface staining with the following fluorescence-labeled antibodies according to the manufacturers´ instructions: HLADR-BV510 (G46-6; BD Bioscience), CD3 APC Cy7 (SK7, BD Bioscience), CD45-FITC (HI30, BD Bioscience), CD14-PECF 594 (MφP9, BD Bioscience), CD19-PE Cy5 (HIB 19, BD Bioscience), CD8-PerCP CY5.5 (RPA-T8, BD Bioscience), CD4-PE Cy7 (RPA-T4, BD Bioscience), and CD56-BV785 (5.1H11, Biolegend). For the lysis of red blood cells, FACS lysing solution (BD Bioscience) was added. Viability dye (VD, eBioscience) staining was used to evaluate apoptotic cells. Cell frequencies were evaluated on the LSR Fortessa cytometer (BD Bioscience, San Jose, CA, USA). Gating strategy for cells is presented in Supplementary Figure S1.

Peripheral blood mononuclear cells (1 × 10⁶) were stimulated with PMA (500 ng/ml), ionomycin (2 μg/ml), and brefeldin A (10 μg/ml) for 5 h at 37°C in 5% CO₂. The surface were stained with CD3-BV421 (UCHT1), CD8-percp-cy5.5 (RPA-T8), and CD4-FITC (RPA-T4) (BD Biosciences), and intracellular staining was performed using anti-IL-17A-PE (N49-653), IL-10-APC (JES3-19F1) (BD Biosciences), IL-4-APC (8D4-8), TGF-β1-PE (TW4-6H10), TNF-α-PE-cy7 (MAb11), and IFN-γ-PE-cy7 (4S.B3) (Biolegend, San Diego, CA, USA) and the foxp3/transcription factor staining buffer set (eBioscience, San Diego, CA, USA) according to the manufacturer’s protocols. Unstimulated cells were used as a negative control.