Absolute γδ T cell counts were performed on 100 μl of fresh PB stained with following anti-human monoclonal antibodies (mAbs): CD3 (SK7; BV605) and CD45 (H130; AF700) (BioLegend) and Vδ2 (IMMU-389; FITC) (Beckman Coulter). We then used CountBright™ Absolute Counting Beads (Invitrogen) according to the manufacturer’s instructions.
For both regular and intracellular staining, γδ T cells were first screened for viability with Zombie Aqua™ Fixable Viability kit (BioLegend) and then processed as previously described [20 (link)]. The following mAbs were used: CD28 (CD28.2; PE-Cy7) (BioLegend); Vδ2 (B6; BUV395), CD3 (UCHT1; BUV661), CD45RA (H100; BUV737), CD16 (348; BUV496) (BD); CD57 (REA769; PE-Vio615) (Miltenyi); CD27 (0322; APCeFluor780) (eBioscience). The intracellular amounts of TNF-α (Mab11; PE) and IFN-γ (B27; Bv711) (BD) as well as the frequency of cytotoxic CD107apos cells (H4A3, PE) (BD Biosciences) was evaluated after stimulating γδ T cells with Phorbol myristate acetate (PMA; 0.5 μg/mL) and Ionomycin (0.1 μg/mL) (Sigma Aldrich).
Flow cytometry experiments were performed on FACS Symphony™ (BD). All data and t-SNE algorithm were analyzed with FlowJo Software (version 9.6) (FlowJo LLC) using single stained controls BD CompBeads™ (BD).
For both regular and intracellular staining, γδ T cells were first screened for viability with Zombie Aqua™ Fixable Viability kit (BioLegend) and then processed as previously described [20 (link)]. The following mAbs were used: CD28 (CD28.2; PE-Cy7) (BioLegend); Vδ2 (B6; BUV395), CD3 (UCHT1; BUV661), CD45RA (H100; BUV737), CD16 (348; BUV496) (BD); CD57 (REA769; PE-Vio615) (Miltenyi); CD27 (0322; APCeFluor780) (eBioscience). The intracellular amounts of TNF-α (Mab11; PE) and IFN-γ (B27; Bv711) (BD) as well as the frequency of cytotoxic CD107apos cells (H4A3, PE) (BD Biosciences) was evaluated after stimulating γδ T cells with Phorbol myristate acetate (PMA; 0.5 μg/mL) and Ionomycin (0.1 μg/mL) (Sigma Aldrich).
Flow cytometry experiments were performed on FACS Symphony™ (BD). All data and t-SNE algorithm were analyzed with FlowJo Software (version 9.6) (FlowJo LLC) using single stained controls BD CompBeads™ (BD).
Free full text: Click here
