Anti-CD4, anti-CD62L, anti-CD44, anti-CD45.1, anti-CD45.2, anti-CD45RB, anti-TCR-β, anti-IL-10Rα (clone: 1B1.3a, PE) and isotype control (rat IgG1,K, PE) were purchased from BioLegend. Anti-STAT3 (pY705) and anti-pp38 MAPK were purchased from BD Biosciences. To identify dead cells, 7-AAD (Biolegend) staining was performed.
Anti-human anti-CD4, anti-CD45RA and anti-CD49b (clone: P1E6-C5) were purchased from BioLegend. Anti-LAG-3 was purchased from eBioscience (clone: 3DS223H). The staining for LAG-3 and CD49b was performed at 37°C for 30 min.
For intracellular pSTAT3 and pp38 MAPK staining, cells were fixed with PhosFlow Lyse/Fix Buffer (BD Bioscience) for 10 min at 37°C and permeabilized with Perm Buffer III (BD Bioscience) for 30 min on ice. The cells were stained for pSTAT3 or pp38 MAPK and extracellular markers for 1 hour at room temperature before they were acquired on a LSRII flow cytometer (BD Bioscience).
Anti-human anti-CD4, anti-CD45RA and anti-CD49b (clone: P1E6-C5) were purchased from BioLegend. Anti-LAG-3 was purchased from eBioscience (clone: 3DS223H). The staining for LAG-3 and CD49b was performed at 37°C for 30 min.
For intracellular pSTAT3 and pp38 MAPK staining, cells were fixed with PhosFlow Lyse/Fix Buffer (BD Bioscience) for 10 min at 37°C and permeabilized with Perm Buffer III (BD Bioscience) for 30 min on ice. The cells were stained for pSTAT3 or pp38 MAPK and extracellular markers for 1 hour at room temperature before they were acquired on a LSRII flow cytometer (BD Bioscience).