Cd4 percp cy5.5 antibodies

PBMCs were counted and 1 × 10⁶ cells were resuspended in flow buffer (PBS+2% FBS, 1 mmol/L EDTA, and 0.1% sodium azide) containing CD3-APC-H7 (clone SK7, BD Biosciences) and CD4-PerCP-Cy5.5 antibodies (clone L200, BioLegend) for 30 minutes at 4⁰C. After washing with flow buffer, cells were permeabilized and fixed using the FOXP3 Fixation/Permeabilization Concentrate and Diluent Kit (eBioscience) according to the manufacturer's instructions. Permeabilized cells were stained with biotinylated anti-human ICOS antibody (clone: M13, Jounce Therapeutics, a noncompetitive binder to ICOS compared with vopratelimab) for 30 minutes at 4°C. After washing, cells were incubated with streptavidin-PE (BioLegend) for 30 minutes at 4°C, washed and then resuspended in flow buffer. In some experiments, Tbet-BV421 (clone 4B10, BioLegend) was added at the intracellular staining step.
Flow cytometry was performed on the same day using the FACSCanto II or LSR Fortessa (BD).
After January 2019, all clinical samples were analyzed at FlowMetric. The same method, flow panel, and analysis were followed at both Jounce and FlowMetric.

For naive CD4 T cell selection, magnetically selected CD4-enriched splenocytes were labeled with CD25-FITC, CD62L-PE, and CD4-PerCP/Cy5.5 antibodies (BioLegend). The cells were then subjected to cell sorting (FACSAria) to obtain CD4⁺CD62L⁺CD25⁻ naive T cells. A cell purity of more than 95% was routinely achieved. For cytokine staining, activated T cells were re-stimulated with PMA (50 ng ml⁻¹) and ionomycin (1 μM) for 4 h and then stained using the Fixation and Permeabilization Solution Kit with BD GolgiStop (BD Biosciences, Franklin Lakes, NJ, USA). For transcription factor staining, the cells were stained with an anti-Foxp3 antibody (eBioscience, Santa Clara, CA, USA) using the Transcription Factor Staining Buffer Set (eBioscience). Occasionally, the FOXP3 Fix/Perm Buffer Set (BioLegend) was used to stain GFP-expressing cells. The cells were then analyzed by flow cytometry (FACSCalibur).