Anti cd45ra bv510

For fluorescence-activated cell sorting (FACS) analysis, cells were labelled with saturating amounts of antibodies in FACS buffer (PBS containing 0.1% BSA and 0.02% NaN₃) to stain cell-surface antigens for 15 min on ice, followed by a fixation/permeabilization step (Fix/Perm buffer, eBioscience) for 30 min at room temperature and intracellular staining for 45 min at room temperature in permeabilization buffer (eBioscience). Stained cells were analysed on an LSR II flow cytometer (BD Biosciences).
For the staining of PBMC, the following Abs were used: Anti-CD3-PE-Cy7, anti-CD4-PerCP, anti-CD4-Pacific Blue, anti-CD4-FITC, anti-CD4-Alexa Fluor 700, anti-CD8-PE, anti-CD25-APC, mIgG₁-APC, anti-CD45RA-PE, anti-CD45RA-PErCP-Cy5.5, anti-CD127-PE, anti-Foxp3-APC anti-Foxp3-Pacific Blue, anti-CCR7-Alexa Fluor 488, anti-Ki-67-Alexa Fluor 700, anti-CTLA-4-PE, anti-CTLA-4-PE-Cy7 (all Biolegend), anti-CD25-PE, anti-CD8-FITC, PE-Cy5-streptavidin, anti-CD86-biotin (all BD Bioscience) and viability dye (Thermo Fischer).
For di-4-ANEPPDHQ (ANE) staining, PBMC were incubated with 4 mM of ANE (Invitrogen) together with anti-CD4 APC-Cy7, anti-CD45RA BV510 and anti-CD25 APC (all from Biolegend) in RPMI medium for 30 min at 37°C and were immediately analysed by FACS.

A whole blood staining protocol was used for the peripheral blood T cell immunophenotyping. Four hundred fifty milliliters of EDTA blood was labeled with anti–CD3-PE.Cy5, anti–CD4-BV711, anti–CD8a-allophycocyanin, anti–CD45RA-BV510, anti–CD27-BV605, anti–CD28-PE/Cy7, anti–CCR7-PE/Cy5.5, and anti-CD25-allophycocyanin/Cy7 Abs (Biolegend) to determine various T cell subpopulations. Abs were used in 1:100 dilutions and incubated at room temperature for 20 min in the dark. All cells were washed with isotonic PBS with 2% FCS and incubated with 2 ml of 0.16 M of NH₄CL at room temperature in the dark for 10 min for red cell lysis. A final wash with PBS and 2% FCS was carried out, and 0.5 ml of 1% paraformaldehyde was added. CountBright Absolute Counting Beads (Life Technologies) were added according to manufacturer’s protocol for accurate cell counting. Compensation was aided by the Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (BD Biosciences) and automatically calculated by BD FACSDiva software version 6.0. Acquisition was performed via BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed via FlowJo version 7.6.5 software.