Immunophenotyping of PBMC Populations

PBMC were washed and maintained in MACS buffer (Miltenyi Biotech, Auburn, CA, USA) during the staining protocol for specific cell markers. Cells were stained first with viability marker according to the manufacturer’s instructions (Thermo Fisher Scientific LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, Eugene, OR, USA) followed by incubation with an FcR block (BD Pharmingen) and, afterward, with specific Abs to the corresponding extracellular markers such as CD3-FITC and CD4-PE-Cy7 (both from BioLegend, San Diego, CA, USA), and CD25-allophycocyanin (eBioscience) were applied. To stain for intracellular markers, the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences) was used as described previously with anti-Foxp3-eFluor 450 Ab (Invitrogen) [17 (link)]. The appropriate isotype control Abs were applied to separate control tubes as described and the staining profile was used to establish the negative gate [17 (link)]. After a final wash, either 10,000 cells/tube or 30,000 cells/tube out of 1 × 10⁶ cells/tube placed in MACS buffer were acquired in logarithmic scales and analyzed with the BD LSR Fortessa flow cytometer. FlowJo software was used for the live cell gating and their further analysis.