Cd45ra pacific blue

Apoptosis was quantified using cellevent caspase 3/7 green detection reagent (ThermoFisher). Cells were incubated with 3 μM of the reagent for 30 min at 37°C. To separate out nTreg, mTreg, and emTreg, cells were blocked with PBS 2% FBS + human IgG 10 μg/ml and stained with the surface antibodies CD45RA-pacific blue, CD95-BV510 (BioLegend), and HLA-DR-APC-Cy7 (BD) for 30 min at room temperature. Then, cells were fixed and permeabilized with FOXP3 perm/fix buffer kit (ThermoFisher) for 1 h at 4°C, followed by intracellular staining with anti-Ki67-PerCP-Cy5.5 (BD), anti-ICOS-BV605, and anti-CTLA4-BV711 (BioLegend). Samples were acquired on a Fortessa flow cytometer (BD). Flow cytometry data were analyzed using the Flowjo software, version 10.6.2 (BD). For some experiments, bulk Treg cells were cultured in X-VIVO media in the presence of caspase 3/7 green reagent and anti-CD45RO (Alexa Fluor 700; BioLegend) and followed for 24 h by time-lapse imaging, using Nikon AXR Inverted Confocal Laser Scanning Microscope (Nikon Corporation, Minato City, Tokyo, Japan).

We performed cellular phenotyping on a FACS Canto II flow cytometer (Becton Dickinson, San Jose, CA, USA). To identify CD4 subsets, we used the following fluorochrome-labeled antibodies for surface staining, according to the manufacturer’s instructions: CD45RA-Pacific Blue and CD31-APC (Biolegend, San Diego, CA, USA), and CD4-APC-H7 and CD45RO-PE (BD Pharmingen, San Diego, CA, USA). Isotype-matched control antibodies were ordered from the same companies. After performing an automated cell count, we incubated 50 µL of blood with erythrocyte-lysing buffer (BD-Pharm Lyse, Frankin Lakes, NJ, USA) at room temperature, centrifuged the solution, and discarded the supernatant. The staining time for surface antibodies or isotypes was 30 min, followed by washing with staining buffer (1 × PBS/5% FBC, both reagents from PAA, Coelbe, Germany). Staining was performed on ice.