Anti cd45ra fitc

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll Paque Plus (GE Healthcare, Boston, MA, USA) gradient cell separation according to manufacturer’s instructions. Red blood cells were lysed using a 1× dilution of BD Pharm lyse (BD Biosciences) in sterile water and incubating the cells for 5–10 min. For total CD4, CD8, and B cell samples, isolated PBMCs were labeled sequentially with anti-human CD4 and CD8 magnetic beads (Miltenyi Biotec, San Diego, CA, USA) and respective fractions were obtained by positive magnetic selection while B cell-enriched fraction was obtained by negative selection of CD4⁺ and CD8⁺ PBMCs. For CD4 and CD8 naïve and memory populations, isolated PBMCs were labeled with mouse anti-human fluorescent antibodies: anti-CD3 PE/Cy7, anti-CD4 PE, anti-CD8 APC, anti-CD45RA FITC, and anti-CCR7 Pacific Blue (eBiosciences, San Diego, CA, USA). Cells were stained for 30 min and then washed and sorted on a BD FACS Aria II cell sorter. Naïve CD4⁺ and CD8⁺ T cells were sorted based on the CD45RA⁺ CCR7⁺ phenotype. With this sorting strategy, the naïve T cell compartment did not include CD45RA⁺ CCR7⁻ cells that correspond to the exhausted effector memory T cell (T_EMRA), which is often expanded in patients with WAS (Table 1). Cell purity was checked after sorting and was consistently >92%.