Luminescence microplate reader

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).