Cell counting kit 8 cck 8

Cell growth curves were assessed by using the Cell Counting Kit-8 (CCK-8) (Abmole, Houston, TX, USA). Cells were seeded at a density of 2000 cells per well in 96-well plates and incubated for 1, 2, and 3 days, respectively. Then, the cells were incubated with CCK-8 solution for 2 h. The optical density (OD) values were measured on the microplate reader (Infinite M200 Pro, Tecan Group, Männedorf, Switzerland) at a wavelength of 450 nm. Similarly, cell proliferation was quantified on day 2 using a 5-bromo-2-deoxyuridine (Brdu) Cell Proliferation Assay Kit (BioVision, Milpitas, CA, USA).

Patulin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chitosan (CTS), 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were obtained from Aladdin Biochemical Technology Co., Ltd. (Beijing, China). Sodium selenite, ascorbic acid (Vitamin C, VC) and chloral hydrate were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Dulbecco’s modified Eagle’s medium and penicillin-streptomycin solution were purchased from BasalMedia Technologies (Shanghai, China). Fetal bovine serum was obtained from Gemini Bio-Products (West Sacramento, CA, USA). A Cell Counting Kit-8 (CCK 8) was purchased from Abmole Bioscience (Houston, TX, USA). A reactive oxygen species (ROS) assay kit was obtainedfrom Beyotime Biotechnology (Shanghai, China). An ELISA Kit for lipopolysaccharides (LPS) were purchased from Xinlebio (Shanghai, China). Malondialdehyde (MDA), protein carbonyls (PC), and activities of antioxidative enzymes detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Yak rumen fibroblasts from the 10th generation were seeded in 96-well plates at a density of 1 × 10⁴ cells/mL. Each treatment group was performed in six replicates, and the viability of one group of cells was assessed daily. Cell viability was measured using a Cell Counting Kit-8 (CCK-8; AbMole, Shanghai, China). To conduct the assay, 10 µL of CCK-8 reagent was added to each well following the instructions of manufacturer [70 ]. The absorbance was measured at 450 nm wavelength in a microplate reader (SpectraMax M2, USA) after 4 h of incubation at 37 °C. Using the incubation time as the abscissa and the average OD value as the ordinate, the cell growth curve was drawn. Yak rumen fibroblasts were seeded in 96-well plates at a density of 1 × 10⁴ cells/mL and challenged with LPS (Solarbio, Beijing, China) at different concentrations (0, 2, 4, 8, 16, and 32 μg/mL) for 24 h. In this study, we selected a range of LPS concentrations to reflect the reported concentrations of rumen LPS in the literature and the experimental concentrations that affect the functionality of fibroblast cells in vitro [71 (link),72 (link)]. The CCK-8 was used to detect the effect of lipopolysaccharide on cell viability.

MLO-Y4s were planted at a density of 1×10⁴ cells/mL (100μL/well) and cultured for 24h for attachment. They were treated with varying concentrations of LDL or ox-LDL (0, 1, 2.5, 5, 10, 25, 50, or 100μg/mL) for 12, 24, or 48h. Cell viability was tested with Cell Counting Kit-8 (CCK-8, AbMole, Shanghai, China). Microplate reader (Thermo Fisher Scientific, Waltham, USA) was used for optical density measurement at 450 nm.

In order to evaluate the viability of the cells after transfection, we conducted cell viability experiments by the Cell Counting Kit-8 (CCK-8) (M4839, AbMole, China). First, the cells were seeded in 96-well plates. When the fusion degree of cells reached about 80%, CCK-8 solution was added to the well and incubated for 2 hours. The absorbance at 450 nm was measured with a microplate reader (SpectraMax5, Molecular Devices, USA).