Cd62l apc cy7

Surface staining (including preincubation with FcR block; anti CD16/32 (BD Biosciences) was performed as described previously [16 (link)]. The following antibodies were used: CD3 Pacific Blue, CD3 PE-Cy7, CD3 FITC, CD8 PerCP-Cy5.5, CD4 V500, CD4 APC, CD44 PE-Cy7, CD62L APC-Cy7, CD69 FITC, CD25 PE (all BD Biosciences). For viability analyses, cells were stained with Annexin V-PE (BD Biosciences) together with antibodies in HBBS/2%FCS (both Biochrom); 7AAD (eBioscience) was added approximately 5 min before the sample was acquired at the flow cytometer. For the staining of intracellular antigens, cells were fixed and subsequently permeabilized to the staining of surface antigens. The FoxP3 PE staining buffer set (eBiosciences) was used for the detection of Foxp3. For IL-2 staining (IL-2 PE, BD Biosciences), fixation buffer and intracellular staining permeabilization wash buffer (Biolegend) were used. Data were acquired on a FACSCalibur (CellQuest, BD Biosciences) or FACSVerse (FACSuite, BD Biosciences) and analyzed with FlowJo software. The concentration of secreted IL-2 in the culture supernatants was measured by Luminex xMAP Technology using a Bio-Plex Pro Mouse Cytokine IL-2 Set (No. 171G5003M), according to the manufacturer’s instructions, on a Bio-Plex suspension array system (all Bio-Rad).