Anti human cd8 apc h7

Sorted CD38⁺ and CD38^- CD8⁺ T cells were plated at 50,000 cells/well in RPMI 1640 containing 25 mM Hepes, 2 mM L-glutamine (Invitrogen), and supplemented with 10 units/mL of Penicillin (Life Technologies), 10 μg/mL of Streptomycin (Life Technologies) and 10% fetal bovine serum (Life Technologies) in a 96-well plate pre-coated overnight with 10 μg/mL of anti-human CD3 OKT3 antibody (Biolegend) together with 0.75x10⁶ cells/mL autologous CD8^- cells, anti-human CD107a-FITC (Biolegend, 1:200 dilution) and 1 μg/mL of co-stimulatory antibodies anti-human CD28 and anti-human CD49d (Becton Dickinson) for 5 hours at 37°C in an atmosphere of 5% C0₂. Following stimulation, cells were resuspended in 20 μl of staining buffer containing anti-human CD4-BV510 (Biolegend, 1:200 dilution), anti-human CD8-APC-H7 (Becton Dickinson, 1:400 dilution) for 20 minutes at 4°C, washed and resuspended in staining buffer before acquisition on LSR Fortessa 4 (Becton Dickinson) with Diva software. FlowJo version 6.0 was used for gating.

Staining buffer was PBS supplemented with 0.5% FCS and 4 mM EDTA. Whole blood collected in Lithium Heparin vacutainers was lysed and fixed with BD FACS lysing solution (Becton Dickinson) and lymphocytes permeabilized with BD FACS permeabilising solution 2 (Becton Dickinson) according to the manufacturer’s instructions. Cells were then resuspended in 50 μl of staining buffer containing anti-human CD4-BV510 (Becton Dickinson, 1:200 dilution), anti-human CD8-APC-H7 (Becton Dickinson, 1:400 dilution), anti-human CD19-PE-Cy7 (Biolegend, 1:200 dilution), anti-human CD38-APC (Biolegend, 1:400 dilution), anti-human Perforin-PE (Biolegend, 1:400 dilution), anti-human Granzyme B-Pacific Blue (Biolegend, 1:400 dilution) and 1 μl of human Fc receptor blocking solution (Human TruStain FcX, Biolegend) for 30 minutes at room temperature, washed and resuspended in staining buffer before acquisition on LSR Fortessa 4 (Becton Dickinson) with Diva software. FlowJo software version 6.0 was used for gating.

CD38⁺ CD8⁺ T cells, CD38^- CD8⁺ T cells as well as CD8^- cells were sorted from freshly isolated PBMC. Approximately 10x10⁶ cells were resuspended in 50 μl of staining buffer containing anti-human CD4-BV510 (Biolegend, 1:200 dilution), anti-human CD8-APC-H7 (Becton Dickinson, 1:400 dilution) and anti-human CD38-PerCpCy5.5 (Biolegend, 1:400 dilution) for 20 minutes at 4°C, washed and resuspended in staining buffer. Just before the sorting, 1 μg/mL of propidium iodide (Sigma-Aldrich) was added to allow for assessment of viability. Pi^-CD8⁺CD4^-CD38⁺, Pi^-CD8⁺CD4^-CD38^-, and Pi^-CD8^- cells were sorted using a BD Aria III cell sorter (Becton Dickinson) directly in staining buffer and kept on ice until further use for in vitro assays.