Dppiv glo protease assay

DPP-IV inhibition was measured as reported previously by Mojica et al. [23 (link)] using the DPP-IVGLO^® Protease Assay (G8351, Promega, Madison, WI, USA). Samples were diluted to a factor of 1:10 to 1:100 with deionized water. In a white-walled 96-well plate, 40 µL of these diluted samples, sitagliptin (positive control, 50–500 nm) or buffer pH 8.0: 100 mM TRIS, 200 mM NaCl, 1 mM EDTA (negative control) were added, followed by the addition of 10 μL of DPP-IV human enzyme (10 ng/mL) and 50 μL of DPP-IVGLO^® reagent. The blank contained only 50 µL of buffer pH 8.0 and 50 µL of DPP-IVGLO^® reagent. Luminescence was measured after mixing and incubating for 30 min at 27 °C using a SpectraMax^® i3 Multi-Mode Microplate Reader (Molecular Devices^®, LLC; San Jose, CA, USA). Results were presented as IC₅₀ values (mg/mL).

In a 96-well, U-bottom plate (NEST, USA), CAR-T cells (effectors) and Nalm6-Luc-GFP (targets) or K562-Luc-GFP (targets) were cultured together at 37°C for 24 h at various effector- to-target ratios (E:T or E/T); the targets were 1 × 10⁴/well. Supernatants were harvested for cytokine secretion detection following the centrifugation of the plate. Avoiding the unequal transduction of CAR-positive in T cells, non-transduced Ctrl-T cells were supplemented to adjust both the number of CAR⁺ T cells, and the total number of T cells remained consistent in all groups. The substrate was added with the DPPIV-Glo™ Protease Assay (Promega, USA) and immediately centrifuged and detected. The results are reported as the percentage of killing based on the luciferase activity in the wells with tumor cells but without T cells [% killing=100−((RLU from well with effector and target cell coculture)/(RLU from well with target cells) ×100)].