We assessed T cell proliferation by measurement of Ki-67 (BD Biosciences) (Figure 1). In brief, 1 £ 10 6 PBMCs were incubated with Pac-Blue-conjugated anti-CD3 (BioLegend), AmCyan-conjugated anti-CD8 (BD Biosciences), and APC-conjugated anti-CD38 (eBioscience), washed twice with FACS buffer, and fixed and permeabilized with ice-cold methanol. Samples were then incubated after the addition of FITC-conjugated anti-Ki-67 (BD Biosciences) and analyzed.
We assessed the potential for granule-mediated cytotoxicity by using granzyme B content as a surrogate (Figure 1). In brief, 1 £ 10 6 PBMCs were incubated with Pac-Blue-conjugated anti-CD3 antibody (BioLegend), AmCyan-conjugated anti-CD8 (BD Biosciences), APC-conjugated anti-CD38 (eBioscience), and FITC-conjugated anti-CD57 (BD Biosciences). Cells were then washed, fixed, and permeabilized with Cytofix/Cytoperm reagent (BD Biosciences) in accordance with the manufacturer's instructions. Samples were then incubated after the addition of PE-conjugated granzyme B (BD Biosciences) and analyzed.
We assessed the potential for granule-mediated cytotoxicity by using granzyme B content as a surrogate (Figure 1). In brief, 1 £ 10 6 PBMCs were incubated with Pac-Blue-conjugated anti-CD3 antibody (BioLegend), AmCyan-conjugated anti-CD8 (BD Biosciences), APC-conjugated anti-CD38 (eBioscience), and FITC-conjugated anti-CD57 (BD Biosciences). Cells were then washed, fixed, and permeabilized with Cytofix/Cytoperm reagent (BD Biosciences) in accordance with the manufacturer's instructions. Samples were then incubated after the addition of PE-conjugated granzyme B (BD Biosciences) and analyzed.