Evaluating CAR Activation in NFAT-GFP Hybridoma Cells

CAR constructs were tested in vitro in a murine NFAT-GFP hybridoma cell line that encoded GFP under the control of an NFAT-dependent IL-2 promoter [32 (link)]. Transduction of the hybridoma cells was performed as described in Section 2.5. HEK293T cells were transfected with the TSPAN7 antigen vector to express the target antigen. TSPAN7 expression was verified with flow cytometry staining with an anti-TSPAN7 Alexa Fluor™ 647-conjugated antibody (FAB9179R, R&D Systems, Wiesbaden, Germany). After 48 h, hybridoma cells were either activated in TSPAN7 protein- or peptide-coated wells, or mixed with antigen-expressing HEK293T cells in 48-well culture plates (677 180, Greiner Bio-One). After 24 h, the cells were harvested and stained with an anti-Fab fragment Alexa Fluor 647-conjugated antibody (109-605-006; Jackson ImmunoResearch, West Grove, PA, USA) for CAR expression and an anti-Her-2 PE-conjugated antibody (324405; BioLegend) for negative gating of HEK293T cells. Cells were analyzed by flow cytometry for the HEK293T marker (PE), CAR expression (Alexa Fluor™ 647), and CAR activation (endogenous GFP).