Cck 8

The cell counting kit-8 assay was used to determine the vitality of the cells (CCK-8, E606335, Sangon Biotech, China). Cells were seeded at a density of 1 × 10⁵ cells per well in 96-well cell culture clusters and cultivated for 1 day, 2 days, and 3 days, respectively. After culture, 10 μL CCK-8 solution was applied to each well, and the absorbance was measured with a microplate reader at 450 nm within 4 hours.

Cells were seeded in a 96-well plate and treated with anlotinib in the presence or absence of bafilomycin for 24 h. After treatment, 10 μl of CCK-8 (cell counting kit 8, E606335-0500; Sangon Biotech, Shanghai) solution was added to each well of the plate and incubated the plate for 1–4 h in the incubator. Finally, the absorbance at 450 nm was measured using a microplate reader.

The viability of HEI-OC1 cells was assayed with a cell counting kit (CCK-8, Sangon Biotech, Shanghai, China). Cells were cultured in 96-well plates at a density of 1 × 10⁴ per well and treated with various concentrations of H₂O₂ (100, 200, 300, 400, 600, and 800 μM) for 1 h, then 10 μL CCK8 solution was added, the solution was incubated at 37 °C for 4 h. The optical density of each well was measured at 450 nm using a FLUO star Omega microplate reader (BMG Labtech GmbH, OFFENBURG, Germany).

Cell viability of RAW264.7 or L929 cultured on scaffolds was determined by CCK‐8 (E606335, Sangon Biotech) according to the manufacturer's protocol. Briefly, different composites (1 mL prior to freeze‐drying; ChCo contained 5 mg chitosan and 5 mg collagen, whereas ChCo‐TAMS containing additional 20 mg PLGA/TA microspheres) were added to 1 mL or 2 mL of culture medium (1:1 Dulbecco's modified Eagle's medium to Ham's F‐12 medium supplemented with 10% FBS, 0.224% NaHCO₃ and 1% penicillin‐streptomycin) after cell seeding at a density of 1 × 10⁵ cells/mL. After culturing for 24 or 48 hours, cell viability was determined at 450 nm using a microplate photometer (Thermo Scientific). Cells were also stained with calcein AM and PI (Life Technology) for 15 minutes at 37°C, rinsed with PBS and then imaged using a fluorescence microscope (Nikon Eclipse Ti‐U, Japan).

After stable transfection, cells were digested with 0.25% trypsin for 1 min and counted by a hemocytometer. Cells (2000 cells/well) were seeded into 96-well plates with 100 μl per well of complete medium. Then, cells were incubated at 37 °C in an environment with 5% CO₂. At the specific time points, before 10 μl CCK-8 (Sangon Biotech, Shanghai, China) solution was added, the medium was replaced by 100 μl fresh culture medium. After incubating 1 h at 37 °C, the absorbance was recorded at 450 nm by using GloMax DISCOVER (Promega, New York, USA). Each sample had three replicates.