Anti human cd25 pe cy7

For the detection of allogeneically activated Tregs multicolor flow cytometry was performed using a combination of following monoclonal antibodies: anti-human CD4 Brilliant Violet 421 (clone: RPA-T4, biolegend), anti-human CD25 PE-Cy7 (clone: M-A251, biolegend), anti-human GARP PE (clone: ME-9FI, biolegend). Intracellular staining of FOXP3 APC (ebioscience, clone: PCH101) was performed according to manufacturer’s instructions. Subsequent flow cytometry was performed on a LSR II (BD Biosciences). Stimulator PBMCs were identified by CFSE labelling and excluded from the analysis. Tregs were identified as CD4⁺CD25^highFOXP3⁺, activated Tregs were identified by their co-expression of GARP. Frequency of activated Tregs was calculated as ratio of CD4⁺CD25^highFOXP3⁺GARP⁺ to CD4⁺CD25^highFOXP3⁺ (Supplementary Fig. 1).

Cytometric analysis was performed according to standard procedures, and samples were acquired on a Navios Flow Cytometer (Beckman Coulter, Brea, CA, USA) followed by data analysis using Kaluza Flow Cytometry Analysis Software (Beckman Coulter) and FlowJo^TM (FlowJo Inc., BD, Franklin, NJ, USA). Cryopreserved PBMCs were thawed and analyzed as follows: A total of 0.5 – 1 million PBMC in 100 µL of complete medium were incubated for 30 minutes at room temperature with 5 µL anti-human CD3-PerCP (BioLegend, San Jose, CA, USA), 5 µL anti-human CD4-FITC (BioLegend), 5 µL anti-human CD25-PECy7 (BioLegend), and 20 µL anti-human CD127-PE (Beckman Coulter) antibodies. After washing with 1X PBS, cells were incubated over night at 4 °C with the 1× FIX/PERM buffer of the internalization kit (eBioscience, Thermofisher) following manufacturer’s instructions. The next day, cells were washed and then incubated for 30 minutes at 4 °C with 5 µL of anti-human FOXP3-APC antibody (eBioscience). After washing with the washing buffer, the PBMCs were analyzed with FACS, with at least 20,000 lymphocyte-gated events acquired for each sample. Mean fluorescence intensity (MFI) was determined using the geometric mean value of the respective marker antibody. The cell populations were analyzed either as % of CD3⁺CD4⁺ (T lymphocytes) or as % of lymphocytes in the live side scatter low gate (Figure 1).