Cd45ra allophycocyanin

Peripheral blood (20 ml) was obtained in a sodium heparin vacuum tube. Subsequently, the blood sample was centrifuged (700 × g for 20 min at 4°C), the supernatant was discarded and the same volume of PBS was then added. The sample was added onto the surface of lymphocyte separation medium, Ficoll-Paque PLUS (GE Healthcare Life Sciences, Little Chalfont, UK), and centrifuged at 700 × g for 20 min at 4°C. PBMCs were collected from the middle layer and suspended at a density of 2×10⁶ cells/ml in RPMI-1640 medium with GlutaMAX (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% heat inactivated fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.). The cells were subsequently stained at 4°C for 30 min with the following fluorescently-labeled antibodies: CD4-fluorescein isothiocyanate (10 µg/ml; 11-0049-41; eBioscience; Thermo Fisher Scientific, Inc.), CD25-PerCP-Cyanine5.5 (1.25 µg/ml; 45-0259-42; eBioscience; Thermo Fisher Scientific, Inc.) and CD45RA-allophycocyanin (20 µl/test; 550855; BD Biosciences, Franklin Lakes, NJ, USA) to sort for nTreg cells (CD4⁺ CD25⁺ CD45RA⁻ T cells) and naïve T cells (CD4⁺ CD25⁻ CD45RA⁺) on a FACS ARIA II cell sorter. Prior to cell staining, Fc receptors were blocked by incubating cells with 10% normal human serum (ImmunoReagents, Inc., Raleigh, NC, USA) for 20 min at 4°C.