Quanti luc 4 reagent

Jurkat TCR-hPD-1 cells were precultured in a medium with or without 300 μM 2FPF for 3 d. Antibodies and PD-1 proteins were diluted in PBS and added to 96-well flat-bottom plates. Jurkat-Lucia TCR-hPD-1 cells and Raji-APC-hPD-L1 were added to the wells according to the manufacturer’s directions, and plates were incubated at 37°C for 6 h. Supernatants were transferred into a 96-well white plate (VWR) and assayed with QUANTI-Luc 4 reagent (InvivoGen). Luminescence was read with an Envision system.

Jurkat-Lucia™ NFAT-CD16 reporter cells was obtained from In vivoGen (In vivoGen, CA, USA) and maintained in RMPI supplemented with 10% FBS, 10 μg/ml blasticidin and 100 μg/ml zeocin. ADCC reporter assay was performed according to the manufacturer’s instructions. In brief, 1x10⁵ tumor cells were incubated with the indicated amount 14F7-IgG1 antibody for 1h at 37°C in a 96-well plate. As a negative control polyclonal human IgG1 antibody (BioXcell, NH, USA) was used. Next, 2x10⁵ Jurkat-Lucia™ NFAT-CD16 were added, and plates was incubated at 37°C. After 18h incubation, QUANTI-Luc™ 4 Reagent (In vivoGen) was added to the plate and immediately read out on a GloMax 96 Microplate Luminometer (Promega, WI, USA). RLU of max was calculated relative to the highest concentration (50 μg/ml) of 14F7-IgG1 antibody.

A Cell Counting Kit-8 (CCK-8, DOJINDO Laboratories, Kumamoto, Japan) assay was performed according to the manufacturer’s recommendations. To measure the binding affinity of PD-L1 for PD-1, Raji-APC-hPD-L1 cells (approximately 5 × 10⁴ cells/mL) were pre-incubated with rapamycin for 6 h and co-cultured with Jurkat-Lucia™ TCR-hPD-1 cells (approximately 1 × 10⁵ cells/mL) in a 96-well plate. After an additional 24 h of incubation, 50 µL of QUANTI‑Luc™ 4 reagent (InvivoGen) was added to each well, and luminescence was immediately measured using a luminescence microplate reader (PerkinElmer, Waltham, Massachusetts, USA). To measure cytolytic activity, SK-Hep1R cells were maintained for 48 h at 37 °C in 12-well plates at an effector cell: target cell (E:T) ratio of 10:1. The cytolytic activity was assessed using crystal violet staining (0.25% v/v in phosphate-buffered saline (PBS)) after fixation with methanol. Positive tumor regions were quantified using cellSens Imaging software (Olympus, Tokyo, Japan), with images acquired using a BX53 light microscope (Olympus). Glucose uptake was determined using a Glucose Assay kit (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. Absorbance was measured at 440 and 640 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).