Sim0006

The functionality of CAR-iMACs was assessed by co-culturing them with luciferase-expressing tumor cells in a 96-well plate (WHB-96-2). CAR-iMACs and 2 × 10³ tumor cells were mixed in a total volume of 100 μL RPMI 1640 complete medium, and the number of cells was determined based on the indicated E:T ratio. After co-culturing for 24 or 48 h, 25 μL of 33 mg/mL D-Luciferin (GoldBio, 115114-35-9) was added to each well, and luminescent signals were analyzed using a microplate reader (TECAN, SPARK).
For the 4-OI supplement assay, iMACs were pre-treated with 4-OI (MCE, HY-112675, 250 μM) or DMSO (Sigma-Aldrich, 41639) control for 3 h before challenging them with LPS (InvivoGen, tlrl-eblps) plus human IFN-γ (PeproTech, 300-02-100UG) (50 ng/mL each). The iMACs were then co-cultured with luciferase-expressing tumor cells.
To test the function of NRF2, SFN (MCE, HY-13755, 10 μM) was added to the co-culture system.
To test the function of cytokines, the supernatant was collected after iMACs were co-cultured with HO-8910 cells for 24 h (E:T = 10:1). Then human IgG1 isotype control (BioXcell, BE0297), neutralizing antibody (10 μg/mL) of IFN-γ (BioXcell, BE0235), or TNF-α (BioXcell, SIM0006) was added to the supernatant, and the cytotoxicity activity was measured by luciferase assay.

Model therapeutic antibodies were IFX biosimilar (SIM0006; BioXcell), Bs-mAb1 and Bs-mAb2 were internally produced at Merck KGaA, trastuzumab IgG1 was made at Merck KGaA. Control-positive proteins were KLH (H7017; Sigma-Aldrich) and Bet v1a (BET_1_1A; Biomay).