Cell counting kit 8 cck 8

DOX (doxorubicin hydrochloride) was obtained from Beijing Ouhe Technology Co., Ltd (Beijing, China), and dimethoxycurcumin (DiMC) was laboratory-made with an HPLC purity >98%. The copolymers of mPEG-PLA with various block length ratios (mPEG1000-PLA2000, mPEG2000-PLA2000, mPEG2000-PLA5000) were supplied by Shanghai Leon Chemical Ltd (Shanghai China). The dialysis tubes with a molecular mass cutoff of 8–14 KDa were purchased from Spectrum Laboratories (Houston, USA). The Cell Counting Kit-8 (CCK-8) was the product of Dalian Meilun Biotech Co., Ltd (Dalian, China), and the kits for biochemical assay were supplied by Nanjing Jiancheng Biochemistry Co. Ltd. (Nanjing, China). All solvents and other reagents were available commercially and of analytical grade or higher. Ultra-pure water prepared by a lab purification system was used throughout the experiment.

Sodium Hyaluronate (MW = 100 kDa) was provided by Shandong Freda Biochem Co., Ltd. (Shandong, China). Dowex^® 50WX8-200, tetrabutylammonium hydroxide (TBA-OH), benzyl bromide, TritonX-100 and hyaluronidase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anhydrous dimethyl sulfoxide (DMSO), ethyl acetate, and Hexafluoroisopropanol (HFIP) were obtained from Aladdin Reagent Company (Shanghai, China). Doxorubicin hydrochloride (Dox•HCl, >99%) and cell counting kit-8 (CCK-8) were brought from Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). Fluorescein diacetate (FDA), propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), and TRITC-phalloidin were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). DMEM medium, RPMI-1640 and fetal bovine serum were provided by Gibco (Cleveland, TN, USA). Penicillin-streptomycin and trypsin were purchased from HyClone (Logan, UT, USA). All other chemicals were used without further purification.

For cell proliferation assay, LM3 and HepG2 cells were seeded in 96-well culture plates (2000 cells per well). The proliferation rate of HCC cells was detected using Cell Counting Kit-8 (CCK8, MA0218, Meilun, Dalian, China) assay at 0 h, 24 h, 48 h, 72 h, and 96 h according to the manufacturer’s instructions. The absorbance of each well was detected at a wavelength of 450 nm (SpectraMax M5, Molecular Devices, US). For clone formation assay, LM3 and HepG2 cells were seeded into 6-well plates (1000 cells per well) and incubated for 14 days, cells were washed with PBS and stained with 0.5% crystal violet. EdU (Ribobio, Guangzhou, China) incorporation assay was performed as we previously reported [54 (link)]. Briefly, cells were culture in 96-well plates at a density of 1000 cells per well. After 24 h, 50 μM EdU was added to each well and incubated for additional 2 h. The cells were fixed with 4% formaldehyde for 15 min and treated with 0.5% Triton X-100 for 20 min. After washing with PBS, 100 μl of 1 × Apollo reaction cocktail was added and incubated for 30 min. After staining with 100 μl of Hoechst 33342 for 30 min, the cells were visualized using EVOS cell image system.

Cell proliferation was determined with the Cell-Counting Kit-8 (CCK-8; Meilunbio, China) following the manufacturer’s instructions. Briefly, BMSCs at passage 2 were seeded at a density of 1 × 10³ cells per well in a 96-well plate at 37 °C with 5% CO₂. Once the cells adhered to the plate, CCK-8 solution was added. After incubation for 1 h at 37 °C, a microplate reader (Thermo Scientific Multiskan FC, USA) was used to examine the absorbance of the solution at 450 nm. Cell proliferation was measured for three consecutive days. All assays were carried out in triplicate. A histogram was made based on the absorbance values.

Cell viability was estimated by the Cell Counting Kit-8 (CCK-8) (Meilunbio catalog no. MA0218-1). Briefly, 10,000 Vero cells were seeded in a 96-well plate, incubated at 37°C overnight, and then administered gradient concentrations of compounds at 37°C for 48 h. The CCK-8 solution was added, followed by an additional 1-h incubation at 37°C. Absorbance was measured at 450 nm. The concentration of each compound necessary to reduce cell viability by 50% (CC₅₀) was calculated by comparing the values with DMSO-treated cells using a sigmoidal nonlinear regression function in GraphPad Prism 8.0 (GraphPad Software, USA).

Cell proliferation was performed by a Cell Counting Kit-8 (CCK-8) (Meilun Biotechnology Co., Ltd., MA0218-5) according to the manufacturer’s protocol. Briefly, the Ishikawa, HEC-1A, and HEC-251 cell lines were trypsinized and seeded into 96-well plates at a density of 1 ×10⁴ each well. After incubating overnight at 37 °C, the cells were treated with a concentration of 40 µM and 80 µM of formononetin for 24 h. The absorbance of each well was determined at 490 nm under a Smart Microplate Reader (USCNK, Wuhan).