Anti cd8 pe cy7 clone sk1

To evaluate intracellular cytokine production, PBMC were stimulated with PepMix peptide pools of spike glycoprotein, NCAP and VME1 (1 μg of each peptide pool/mL) overnight at 37°C, 5% CO₂ in the presence of brefeldin A at 10 μg/mL (BD Biosciences). PBMC cultured with medium alone or PMA/IONO were used as negative and positive controls, respectively. After the stimulation, PBMC were washed and stained for 20 min at 4°C in PBS with antibodies following an additional staining with Fixable viability Dye (FvD)-eFluor 506 (eBioscience) for 10 min at 4°C. T cell phenotype was investigated performing surface staining with anti-CD3-Pacific Blue (clone UCHT1; BD), anti-CD4-Alexa Fluor 488 (clone RPA-T4; Biolegend), anti-CD8-PE-Cy7 (clone SK1; Biolegend), antibodies. Then, PBMC were permeabilized and fixed with the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Biosciences). Intracellular staining was performed using anti-IFN-γ APC (clone 4S.B3; Biolegend), anti- TNF-α APC AF700 (clone IPM2, Beckman Coulter), anti-IL-2 PE (clone MQ1-17H12, Biolegend) antibodies for 30 min at 4°C. Samples were directly acquired on a Cytoflex (Beckman Coulter) and analyzed with Kaluza software.