Cell counting kit 8 cck

Cell survival rates were assessed using a CCK8 Cell Counting Kit (Beyotime, China), according to the manufacturer’s instructions. Cells were either transfected with the BCL2 overexpression plasmid or with EGFP-N1 as a control (Table S1). Cells were seeded in flat-bottomed 96-well plates at 1 × 10⁴ cells/well and incubated in MEM/EBSS (HyClone, Thermo scientific, USA) medium containing 10% FBS for 24 hours in 37 °C in a humidified incubator with a mixture of 5% CO₂ and 95% air. Each group comprised 8 wells. Subsequently, the cells were transferred to MEM/EBSS medium without FBS and cultured for an additional 24 hours to induce apoptosis. A total of 10 μL of CCK-8 was added to each well, and 100 μl of MEM/EBSS containing 10% FBS and antibiotic without any CCK8 was used as photometric blank. After incubation at 37 °C for 2 hours, the absorbance at 450 nm of each well was determined using a microplate reader (Molecular Devices, USA), and the survival rate was calculated according to the CCK8 kit instructions.

As necroptosis is a type of cell death, cell viability and cytotoxicity can reflect its occurrence in vitro. Cells were cultured in 96-well plates. The CCK-8 cell counting kit (Beyotime, Shanghai, China) was used to evaluate cell viability according to the manufacturer's instructions. Twenty microliters of CCK-8 solution was added to 200 μl of cell culture medium, after which the plates were incubated at 37°C for 2 h. Then, the absorbance at 450 nm was measured. Cytotoxicity was evaluated using an LDH lactate dehydrogenase cytotoxicity test kit (Beyotime). Briefly, cells were cultured in serum-free medium for 24 h. Then, the plates were centrifuged at 400 × g for 5 min. One hundred twenty microliters of supernatant of each well was transferred to a new 96-well plate, and the absorbance at 490 nm was measured immediately.

Cell proliferation was assessed using a CCK-8 Cell Counting kit (Beyotime, Shanghai, China). FBCs were seeded in a 96-well plate (2 × 10³ cells per well) and then cultured for 12, 24, 36, 48, 60, or 72 h before adding 10 μl of CCK-8 (5 mg/ml) to the culture medium in each well. After incubating for 2 h, absorbance at 450 nm was measured using a Thermo-max microplate (ThermoFisher, Massachusetts, United States) reader. All experiments involving each transfection were performed in triplicate. HPPCs were transferred to culture medium with 50 µM 5-ethynyl-20-deoxyuridine (Edu, Beyotime, Beijing, China) for 2 h at 37°C after 36 h transfection. Afterwards, cells were fixed in 4% paraformaldehyde for 15 min at room temperature (RT), and then permeabilized with 0.3% Triton X-100 for 10 min. To block non-specific binding, cells were incubated in blocking buffer (PBS containing 3% bovine serum albumin, 0.3% Triton X-100) for 1 h at room temperature. Next, the cells were incubated with a solution containing 10 mM Edu for 30 min in the dark. Nuclei were stained with 10 μg/ml 4, 6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China) solution in the dark for 10 min. A Leica SP8 confocal microscope was used to capture three randomly selected fields to visualize the number of Edu-stained cells.