Anti human cd19 apc

T cells were stained with anti-hCD45RA-PE (1:400) or anti-hCD45RA.FITC (1:400), anti-hCD62L-PE-Cy7 (1:400), and anti-hCD3-PerCP-Cy5 (1:400) (all from eBioscience-Invitrogen) during 30 min on ice. CAR expression was measured indirectly by the surface expression of truncated EGFR with anti-hEGFR-PE (Biolegend, 1:100). Then, cells were washed with PBS at 400 g for 5 min. Fluorescence was immediately acquired in a FACSCantoII cytometer, and data were analyzed with FlowJo v10 (TreeStart).
In the case of humanized murine samples, cells were washed with cold PBS + 2% BSA + 2 mM EDTA (“FACS buffer”) and incubated with anti-murine CD16/CD32 (Thermo Fisher, 1:100), human FcR blocking (Miltenyi, 1:00), and 5% of mouse serum for 20 min on ice. After one wash with FACs buffer, cells were stained with anti-human CD3-PerCP-Cy5.5 (1:200) for Jurkat-mice model or with anti-human CD19-APC (1:200, eBiosciences) for Namalwa mice model during 30 min on ice and dark for 30 min. Cells were fixed with fresh PFA (2%) prior to acquisition.

For IS formation, the protocol is based on a classical assay previously reported for IS formation in vitro (Roumier et al., 2001). In brief, Raji cells used as APC were preincubated at 37°C for 30 min with either 10 μg/ml of SEE (Toxin Technology, FL, USA) for conjugate formation with Jurkat cells, or with a mix of SAgs composed of SEA, SEC1, SEC3, SEE, and TSST‐1 (10 μg/ml each) for conjugate formation with T lymphoblasts. Conjugates were formed in suspension by incubating SAg‐pulsed Raji cells with Jurkat cells or T blasts, previously infected as described above at 37°C for 20 min, prior to fixation for flow cytometry analysis and immunofluorescence microscopy or lysis for western blotting. Non‐infected cells were used as control. For confocal microscopy experiments, Raji cells were preincubated either with CellTrace Far Red DDAO‐SE or CellTracker Blue CDMC dyes (ThermoFisher) according to the manufacturer's instructions. For flow cytometry experiments, conjugates were stained with anti‐human‐CD3‐FITC (Clone OKT3, ThermoFisher) and anti‐human‐CD19‐APC (Clone HIB19, Invitrogen). Acquisition was performed on a FACSCantoII flow cytometer (BD Bioscience) equipped with 405‐, 488‐, and 633‐nm lasers, or an AttuneNxT flow Cytometer (ThermoFisher) equipped with 405‐, 488‐, 561‐, and 638‐nm lasers. The software Flowjo version 10.4.2 was used for subsequent analysis.