Cd3 pe cy5 clone hit3a

Autoreactive-T cell clones isolated from T1D patients were resuspended in 200 µl of T cell medium (RPMI-1640 (Gibco) +10% pooled human serum +1% penicillin-streptomycin), and stimulated with 50 ng/mL phorbol 12-myristate 13-acetate and 1 µg/mL ionomycin in the presence of 10 µg/mL Brefeldin A for 4 hours at 37°C. After incubation cells were stained with surface antibodies including CD3 PE-Cy5 (clone HIT3a, BioLegend) and CD4 v500 (clone RPA-T4, BD Biosciences) as well as Fixable Viability Stain 450 (BD Horizon). Cells were then fixed and permeabilized as per the manufacturer's instructions (eBioscience). Cells were next stained with antibodies against IFN-γ AF700 (clone 4S.B3, BioLegend), IL-10 PE Cy7 (clone MQ1-17H12, BioLegend), IL-17A APC Cy7 (clone BL168, BioLegend), and IL-4 FITC (clone 8D4-8, eBioscience) for 20 minutes at 4°C. Cells were then washed in PBS and immediately analyzed by flow cytometry on a BD LSRII multi-color flow cytometer. Clones were considered cytokine positive if more than 10% of the cells produced that particular cytokine.

Expanded T cell cultures were rested for 3 days, resuspended in 200 µl of T cell medium, stained with 2.5 uL of each appropriate tetramer, and incubated at 37°C for 30 min, and then activated with 50 ng/mL phorbol 12-myristate 13-acetate and 1 µg/mL ionomycin in the presence of 10 µg/mL Brefeldin A for 4 hours at 37°C. After activation, cells were stained with CD3 PE-Cy5 (clone HIT3a, BioLegend) and CD4 v500 (clone RPA-T4, BD Biosciences) as well as Fixable Viability Stain 450 (BD Horizon). Cells were then fixed and permeabilized as per the manufacturer’s instructions (eBioscience). Permeabilized cells were then stained with antibodies against IFN-γ AF700 (clone 4S.B3, BioLegend), IL-10 APC H7A (clone MQ1-17H12, BioLegend), IL-17A PE Cy7 (clone BL168, BioLegend), and IL-4 FITC (clone 8D4-8, eBioscience) for 20 minutes at 4°C. Cells were then washed in PBS and immediately analyzed by flow cytometry on a BD LSRII multi-color flow cytometer and analyzed using FlowJo (Treestar Inc). Clones were considered cytokine positive if more than 10% of the cells produced that particular cytokine.