Quanti luc gold substrate

Reporter cell line Jurkat-NFAT-Lucia (Invivogen, France) expressing CD19-CAR (Jurkat CD19-CAR) or IL13-CAR (Jurkat IL13-CAR) were obtained by lentiviral transduction as described before. For activation assay 5x10 4 Jurkat CAR-T cells were mixed with freshly isolated antigen-positive or antigen-negative AVs at CAR-T cell-to-AVs cell ratios (0, 4:1, 2:1, 1:1, 1:2, 1:5, 1:10 or 1:20) in a 96-well plate and incubated for 24 hours at 37°C. 25µl of supernatant was separated from cells by centrifugation (4°C, 300g, 5 min) and transferred to the opaque 96-well plate. Activation of reporter Jurkat-NFAT-Lucia CAR-T cells was measured by level of luciferase activity following reaction with Quanti-luc Gold substrate (Invivogen, France) according to the manufacturer's instructions.
T cell degranulation assay: IL13-CAR T cells were resuspended in TexMACS media in concentration 2x10 6 cells/ml. 100 µl of cell suspension was transferred to the 96-well plate and 2µl of anti-human CD107a antibody (BD) were added in each well. CAR-T cells were mixed with either antigen-positive or antigen-negative AVs at ratio 1:1, or kept untreated (ctl) and were incubated for 2 hours on 4°C. Cell suspension was washed twice with TexMACS media (4°C, 300g, 5 min), stained with anti-human CD3/CD4/CD8 antibodies and analyzed by flow cytometry.