Cell counting kit 8 cck 8 kit

Several HCC transiently transfected cells were seeded in 96 wells (0.5 ~ 1 × 10⁴/well) and the experiments were repeated 3 times for each sample. The cells were incubated with gradient concentrations of sorafenib for 48–72 h. Then the Cell Counting Kit-8 (CCK-8) Kit (Beyotime, China) was used to assess cell viability.

To observe the morphology of ASCs, cells were pre-seeded on the cover glass and transfection was performed. Cells were fixed with 2% glutaraldehyde overnight at 4°C 1 day post transfection. Following dehydration in a series of graded ethanol, the specimens were sputter coated with platinum and observed by scanning electron microscopy (SEM; S-4800; Hitachi, Tokyo, Japan). For proliferation analysis, the ASCs were seeded into a 96-well plate and transfected. The cell proliferation was represented by the cell viability, which was determined using a Cell Counting kit-8 (CCK-8) kit (Beyotime Institute of Biotechnology) continuously for 7 days. Briefly, the medium was removed and replaced with 100 µl reaction solution (CCK-8: medium=1:10). Following incubation for 3 h, the supernatant was transferred into a new 96-well plate and the absorbance was read at 465 nm using a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).

The cell counting kit-8 (CCK-8) kit (Beyotime) and Cell-Light™ EdU Apollo^®488 In Vitro Imaging Kit (RIBOBIO, Guangzhou, China) were used for HCC cell proliferation determination as previously described (Shi et al., 2021 (link)). For colony formation assay, the logarithmic growth cells (1 × 10³) were seeded into 6-well plates and were grown for a period of 2 weeks. Cell colonies were stabilized with 4% paraformaldehyde, stained with 0.1% crystal violet solution and manually counted.

I. obliquus was obtained from the Microbial Genetic Stock Center of Huazhong Agricultural University (Wuhan, China). DEAE cellulose-52, Sephadex G-100, and T-series dextran molecular weight standards (T-10, T-40, T-70, T-500, T-2000) were from Pharmacia (Sweden). D-glucose (Glc), D-mannose (Man), D-arabinose (Ara), D-xylose (Xyl), D-fucose (Fuc), L-rhamnose (Rha), inositol, and erythritol (purity of standards ≥99%) were from Sigma-Aldrich (USA). DMEM, RPMI-1640, trypsin, penicillin-streptomycin, and diethyl pyrocarbonate (DEPC)-treated water were from Gibco (USA). High-Capacity cDNA Reverse Transcription Kit was from Thermo Fisher Scientific (USA). HiPure Fungal RNA Mini Kit was from Megen (China). HiScript II Q RT SuperMix for qPCR Kit was from Vazyme (China). Cell Counting Kit-8 (CCK-8 Kit) was from Beyotime Institute of Biotechnology (China). Concanavalin A (ConA) and camptothecin (CPT) were from Macklin Biochemical Co.(China) Macrophage RAW264.7, human cervical cancer HeLa, and mouse sarcoma S180 cell lines were from American Type Culture Collection (ATCC; USA). RAW264.7 was cultured with 10% FBS and 1% double-strength RPMI-1640, and HeLa and S180 were cultured with 10% FBS and 1% DMEM, for 2 days at 37°C in 5% CO₂ atmosphere. Other reagents were from Sinopharm Chemical Reagent Co. (China).