Anti cd4 fitc clone rpa t4

CD4 T cell clones were seeded in a 96-well V bottom plate and subjected to standard or optimized multimer staining procedures and put in culture in a round bottom 96-well plate in R8 media containing 100 IU/mL of hrIL-2. At time points 6 hour, 24 hours and 48 hours, the cells were collected and stained for viability markers. Cells were washed with PBS and resuspended in 25 µL of PBS containing anti-CD3 AF700 (clone HIT3a, Biolegend), anti-CD4 FITC (clone RPA-T4, Biolegend) and LIVE/DEAD fixable dead cell stain (Vivid, Invitrogen) diluted 1:800 in PBS. The cells were incubated at RT for 30 min and washed with PBS. They were resuspended in 25 µL of AnnexinV buffer 1X with AnnexinV-PE (Becton Dickinson) and incubated for 20 min at 4°C. Cells were then washed and resuspended in AnnexinV buffer 1X and analyzed with the CytoFLEX S Flow Cytometry Analyzer (Becton Dickinson).

For the evaluation of the secreted cytokines on specific stimulation, CD4 T cell clones were plated in a 96-well U-shaped plate, left unstimulated, stimulated with 5 µM of specific HA_307-319 or NY-ESO-1_87-99 peptides or with 5 µM of an irrelevant peptide. The cells were incubated O/N with the addition of 2.5 µg/mL of Brefeldin A. The cells were then collected and transferred to a 96-well V-shaped plate for flow cytometry staining. The cells were first incubated for 30 min at RT with anti-CD3 BV421 (clone UCHT1, Biolegend), anti-CD4 FITC (clone RPA-T4, Biolegend) and LIVE/DEAD fixable dead cell stain (Vivid, Invitrogen) diluted 1:800 in PBS. Cells were then fixed and permeabilized following the recommendations provided with the eBioscience Foxp3/Transcription Factor Staining Buffer Set. Cells were washed twice with the permeabilization buffer 1X provided with the kit and then incubated for a period of 30 min at RT with the anti-IFNγ PE (clone B27, Biolegend) and the anti-TNFα AF700 (clone Mab11, Becton Dickinson) diluted in permeabilization buffer 1X. The cells were washed and analyzed with the CytoFLEX S Flow Cytometry Analyzer (Becton Dickinson).