Anti cd3 pacific blue clone ucht1

PBMCs were stained with anti-CD3 Pacific Blue (clone UCHT1, BD Biosciences), anti-CD56 PE-Cy7 (clone B159, BD Biosciences) for 30 min at room temperature. CD56+ T cells, CD56+ NK cells and CD56− T cells were sorted by BD FACS AriaIII (BD Biosciences, San Jose, CA) and only cells with purity >95% were used in subsequent experiments.

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.

Human peripheral invariant NKT (iNKT) cells were characterized as CD3+ T cells expressing a specific TCR (Vα24-Jα18-Vβ11). To identify iNKT cells, PBMCs were surface-stained with anti-CD3 Pacific Blue (clone UCHT1, BD Biosciences) and anti-Vα24-Jα18 FITC (clone 6B11, BD Biosciences). CD3+ Vα24-Jα18+ double positive cells were defined as iNKT cells. To detect CD16, CD161 and CD69 expression on iNKT and CD56+ T cells, PBMC were surface-stained with anti-CD3 Pacific Blue, anti-Vα24-Jα18 FITC (clone 6B11), anti-CD56 PE-Cy7 (clone B159), anti-CD16 (clone eBioCB16, eBioscience), anti-CD161 (clone DX12, BD Biosciences) and anti-CD69 (clone FN50, BD Biosciences). Samples were analyzed on BD FACS Fortessa (BD Biosciences, San Jose, CA, USA) and the frequencies of CD16+ or CD32+ cells in CD56+ T cells, CD56+ NK cells and CD56− T cells were calculated and compared.