Xfp cell mito stress kit

Mitochondrial function of the CAR-T cells was analyzed with an extracellular flux analyzer XFp (Agilent Technologies, Santa Clara, CA). Each well of the cell culture microplate was coated with CellTak (BD Biosciences), according to the manufacturer’s instructions. To assay mitochondrial function, the sorted CAR-T cells without further culture were resuspended in XF RPMI medium supplemented with 5.5 mM Glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate, and they were seeded at 3 × 10⁵ cells/well. The plate was centrifuged at 200 × g for 1 min and incubated at 37°C in a non-CO₂ incubator for 30–60 min. During incubation, the instrument XFp and its assay cartridges were calibrated according to the manufacturer’s instructions. OCRs were measured under basal conditions and following treatment with 1 μM oligomycin (an inhibitor of ATP synthase), 1 μM FCCP (uncoupling of oxygen consumption from ATP production), and 1 μM rotenone and antimycin A (inhibitor for complexes I and III of the electron transport chain, respectively) (XFp Cell Mito Stress Kit, Agilent Technologies). Four measurements of basal condition and after each treatment were performed. ATP-linked respiration was defined as (last rate measurement before oligomycin addition) − (minimum rate measurement after oligomycin addition). Maximal OCR was measured after FCCP treatment.