Cell counting kit 8 cck 8

Human thyroid papillary carcinoma cell Papillary cells of human thyroid carcinoma BCPAP cell lines (BCPAP) was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Quercetin (CAS No. 117-39-5), luteolin (CAS No. 491-70-3), beta-sitosterol (β-sitosterol) (CAS No. 83-46-5), kaempferol (CAS No. 520-18-3) were purchased from Selleck China company respectively. One thousand six hundred-forty culture medium, dimethyl sulfoxide (DMSO), fetal bovine serum, penicillin streptomycin double antibody, 0.25% trypsin, cell counting kit 8 (CCK8), and DMSO purchased from Sangon Biotech (Shanghai) Co., Ltd. RIPA Lysis Buffer, rabbit derived vascular endothelial growth factor A (VEGFA) antibody, tumor protein p53 (TP53) antibody, transcription factor AP-1 (JUN) antibody, prostaglandin endoperoxidase 2 (PTGS2) antibody, interleukin 6 (IL6) antibody, IL-1B antibody, rabbit derived House keeping protein. (glyceraldehyde-3-phosphate dehydrogenase) antibody and goat anti-rabbit IgG were purchased from Beyotime Biotechnology Co., Ltd. and ThermoFisher Scientific Co., Ltd. respectively. The instrument includes BioRad full-automatic microplate reader, BioRad chemiluminescence gel imaging system, OLYMPUS IX51 inverted microscope, MCO-15AC SANYO CO₂ constant temperature incubator, Eppendorf 5702R low-speed centrifuge.

Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8, Sangon, Shanghai, China). SLF-3D and ULA-3D spheroids (which were dissociated into single cells with Accutase) were plated into a 96-well microplate with 10% FBS DMEM/F12 medium at a density of 1,000 cells per well. At 24, 48, 72, and 96 h, CCK-8 reagent (10 µl per well) was added to the cells and then incubated at 37°C for 2 h, and then the growth curves of cells were generated using absorbance values detected using a microplate spectrophotometer (Tecan, Switzerland) at 450 nm.
To count the cells, the living and dead cells of SLF-3D and ULA-3D spheroids (which were dissociated into single cells with Accutase) were counted using either the Countess^® Automated Cell Counter (Invitrogen) or hemocytometer and using Trypan blue (Invitrogen) exclusion.

Cell viability was determined by a Cell Counting Kit-8 (CCK-8) (Sangon, Shanghai, China). In brief, 7 × 10³ cells were cultured in each 96-well plate, and incubated for 24, 48, 72, or 96 h, respectively. Subsequently, 10% CCK-8 reagent was added to each well for an additional 1 h before the endpoint of incubation at 37 °C in dark. The absorbance was measured at 450 nm (A₄₅₀) by a microplate reader (Thermo Fisher). Experiments were repeated at least three times in 6 replicate wells per sample.

The viability of hBMSCs and chondrocytes was determined using the Cell Counting Kit-8 (CCK-8, Sangon Biotech, Shanghai, China). Cells were seeded in 96-well plates in 5% CO₂ at 37 °C; CCK-8 solution (10 μL) was added to each well and cultivated for 1 h. Next, a microplate reader (BioTek Instruments Inc.) was used to measure the absorbance at 450 nm.