Anti cd3 pe dazzle 594

PBMCs were thawed, washed with PBS, and stained with the Zombie Yellow Fixable Viability Kit for 25 min at room temperature. Subsequently, the cells were washed with PBS and incubated for 25 min at 4°C with antibodies for membrane marker staining prediluted in flow cytometry buffer: anti-CD3 PE/Dazzle 594, anti-CD4 FITC, anti-CD45RA PE/Cy7, anti-CD27 Brilliant Violet 421, anti-inducible T-cell costimulator (ICOS) Brilliant Violet 421, anti-CD127 Brilliant Violet 510, anti-C-C chemokine receptor type 7 (CCR7) PE/Cy7, anti-HLA-DR PerCP, anti-CD62L PerCP/Cyanine 5.5 (all from Biolegend), and anti-CD25 PE (Miltenyi Biotec). Afterwards, the cells were washed with flow cytometry buffer and fixation/permeabilization reagent (Foxp3/transcription factor buffer set, eBioscience) was added to each sample followed by an incubation step of 25 min at 4°C. Then, the cells were washed with permeabilization buffer (Foxp3/transcription factor buffer set) and incubated with anti-Foxp3 APC (eBioscience), prediluted in permeabilization buffer, for 25 min at 4°C. Finally, cells were washed with permeabilization buffer and resuspended in flow cytometry buffer. Acquisition and compensation was performed as described for ICS.

Stimulated PBMCs were stained with MART-1 Tetramer (TCMetrix, Lausanne, Switzerland) PE, anti-CD3 PE/Dazzle 594, anti-CD8 AF700, anti-CD45RA FITC, anti-CD4 PerCP/Cy5.5, anti-CD25 BV605, anti-CD127 APC/Cy7, anti-CD39 APC, anti-CD73 PE/Cy7 (all antibodies from Biolegend, San Diego, CA, USA). Dead cells were excluded using Zombie Aqua (Biolegend, San Diego, CA, USA). Briefly, cells were resuspended and washed in PBS supplemented with 2% FBS (Gibco by Life Technologies, Grand Island, NY, USA), incubated for 45 min at room temperature in the presence of MART-1 tetramer, washed twice and incubated with the indicated antibodies for 20 min at 4 °C, cells were finally washed and analyzed by flow cytometry with a Fortessa instrument (BD Biosciences, San Jose, CA, USA).