Cd45ra v450

We performed immunophenotyping on whole-blood EDTA samples while cell sorting was performed on peripheral blood mononuclear cells (PBMCs) isolated from the whole blood by density gradient centrifugation using LymphoPrep (Sigma) after dextran sedimentation. For sorting, PBMCs were thawed in RPMI (10% FCS) pre-warmed with DNase 200 μl ml^-1 (also used TexMACS 5% human AB serum). Cells were then washed, counted and stained on ice in 100 μl of PBS for every 5 × 10⁶ cells. First, Fc block (BD 564220, 5 μl) was applied for 10 min and, without washing, an anti-idiotype CD19 CAT19 B12RIgG2a (Evitria) (2.5 μl or 3.5 μg) was added for 20 min. Then cells were washed and stained for 30 min with secondary antibody mix containing anti-rat IgG PE for CAR detection (Biolegend 405406, 0.5 μl), CD3 APC-Cy7 (Biolegend 300426, 5 μl), 7-AAD (BD 555816, 5 μl), CD95 BV711 (BD 563132, 5 μl), CD45RA v450 (BD 8053598, 2 μl) and CD62L APC (Biolegend 304810, 3 μl). Cells were washed, resuspended in sorting buffer (PBS (1% FBS) + 1 mM EDTA), passed through a cell strainer (30–40 μM) and then sorted using a FASCAriaIII cell sorter. Raw FACS data were collected using DIVA software (BD Biosciences) and analyzed with FlowJo (TreeStar). When feasible, an aliquot of the sorted cells was re-run through the cell sorter to check fraction purity.