Anti ki67 percp cy5

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).

For proliferation assay, PBMCs were cultivated as described above. Six days after culture, cells were stained for surface markers (anti-CD3-FITC (eBioscience, San Diego, CA), anti-CD4-PE, and anti-CD8-APC (BD Pharmingen, San Diego, CA)) and intracellularly with anti-Ki-67-PercP-Cy5.5 (BD Pharmingen, San Diego, CA), a cellular marker for proliferation [25 (link)]. Cells were then fixed and acquisition and data analysis were performed as described above.