Alexa fluor 647 conjugated anti foxp3 antibody

Total T regs (CD4 + CD25 + FoxP3 high cells) and activated T regs (CD4 + CD25 + FoxP3 high CD45RA -cells) were analyzed by four-color flow cytometric analysis. We used fluorescein isothiocyanate (FITC)-conjugated anti-CD4 antibody (cat. no. 555346; BD Pharmingen, San Jose, CA, USA), phycoerythrin (PE) conjugated anti-CD25 antibody (cat. no. 555432; BD Pharmingen), Alexa Fluor 647-conjugated anti-FoxP3 antibody (cat. no. 560045; BD Pharmingen) and VioBlue-conjugated anti-CD45RA antibody (cat. no. 130-113-360; Miltenyi Biotec, San Diego, CA, USA) for cell staining. One million peripheral blood mononuclear cells (PBMCs) were initially stained with the FITC-CD4 antibody, phycoerythrin (PE)-CD25 antibody and V450-CD45RA antibody for 20 min. Subsequently, intracellular detection of FoxP3 was performed on fixed and permeabilized cells using the human FoxP3 Buffer Set (cat. no. 560098; BD Pharmingen), according to the protocol provided by the manufacturer. After staining with Alexa Fluor 647-conjugated anti-FoxP3 antibody for 20 min, PBMCs were washed twice with PBS containing 2% fetal bovine serum (FBS). More than 20 000 CD4 + T cell events were analyzed using a FACSVerse flow cytometer and the FACSuite software (BD Biosciences, San Jose, CA, USA).