Anti v5 fitc

Protein binding to A549 was tested by flow cytometry. A549 cells were detached with StemPro Accutase^TM Cell Dissociation Reagent (A1110501, Gibco). Cells were incubated with a twofold dilution of V5-tagged WT or mutant rVAR2 (400–12.5 nM) for 30 min at 4 °C, followed by an incubation with 500x diluted Anti-V5 FITC (Invitrogen 46-0308) for detection. Gating strategy showing forward and side-scatter is shown in Fig S12. Samples were processed on an LSR II flow cytometer (BD) and results were analysed using FlowJo^TM software (BD Life Sciences).

To determine target-specific proliferation, 1 × 10⁵ CAR T cells were labeled with 1 µM CellTrace^TM Violet (Life Technology) and co-incubated with either 5 × 10⁵ target cells or 5 × 10⁴ human T-activator CD3/CD28 beads in a final volume of 1 mL RPMI-1640 media supplemented by 10% Hyclone FBS, for 7 days at 37 °C. Cells were then recovered and labeled with anti-human CD3-BV421 (1:100; Biolegend) and anti-V5-FITC (1:200; Invitrogen). After staining, 10 µL of CountBright beads (Thermo Fisher Scientific) were added to the cells in 190 µL volume. The number of cells and beads were calculated following the manufacturer’s protocol.
To measure CD69 expression, cells were pelleted after a 24-h co-culture (E:T = 1:2) for flow cytometry analysis, while supernatants were collected for cytokine analysis. Cells were collected and labeled with anti-human CD69-BV605 (1:100; Biolegend). All flow cytometric analysis was done on an LSRII cytometer (BD Biosciences) using FACSDiva (BD Biosciences) and analyzed with FlowJo software version 10 (Tree Star). Cytokine production was quantified by MSD U-PLEX assays (Mesoscale Discovery) according to the manufacturer’s protocol.