Epoch multifunction microplate reader

The vitality of organoids treated with drugs was determined using the CCK-8 assay. In brief, the dissociated organoids (3000 cells/well) were grown for 3 days in a 5% Matrigel precoated 96-well plate. On day 4, organoids were treated with drugs at various concentrations (0.1–100 uM). The CCK-8 solution was then added and incubated in a CO₂ incubator for 4 hours. The light absorbance at 450 nm was determined using an Epoch multifunction microplate reader (Biotek, USA).

Utilize the Cell Counting Kit-8 kit for assessing cell viability. Inoculate 100 μL of processed 143B and SAOS2 cells into aseptic 96-well plates (1.0 × 10⁴ cells/well) and incubate them in a CO₂ incubator at 37 °C with 5% CO₂ and full humidity. Start drug treatment at concentrations of 0, 12.5, 25, 50, 100, and 200 mg/mL after 24 h of seeding the cells. Following 12, 24, and 48 h of therapy, discard the liquid above and introduce 100 μL of DMEM solution with 10% CCK-8 into every well. Place in the incubator for another hour, then use an EPOCH multi-function microplate reader (BioTek, USA) to determine the absorbance at 450 nm. Construct a dose–response curve and calculate the IC₅₀ value of TGT against osteosarcoma inhibition. Expose the cells to 50 and 75 mg/mL concentrations, either with or without the autophagy inhibitor 3-MA and the apoptosis inhibitor Z-VAD-FMK, for 48 h under identical circumstances. Subsequently, reevaluate the absorbance. Using an ECLIPSE TS2 inverted fluorescence microscope (Nikon, Japan), capture images of the cells treated with TGT (0, 25, 50, and 75 mg/mL) for 48 h to observe their morphological changes.