Cck 8 cell proliferation and cytotoxicity assay kit

THP-1 cells (China Cell Line Bank, Beijing, China) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), and 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. In all experiments, the cells were allowed to acclimate for 24 h prior to any treatment. Analysis was conducted in accordance with a previous study [60 (link)]. The viability of THP-1 macrophages following the various treatments was measured using a CCK-8 cell proliferation and cytotoxicity assay kit (CA1210; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Briefly, THP-1 cells were seeded in 10% FBS-RPMI at a density of 5 × 10⁴ cells/100 μL in 96-well plates, after which the cells were treated with PMA (100 ng/mL) and EEAR (0, 12.5, 25, 50, 100, 200 μg/mL) for 24 h. A 10 µL CCK-8 solution was added to each well and incubated at 37 °C for 1.5 h, in accordance with the manufacturer’s instructions, after which the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 medium, fetal bovine serum, penicillin/streptomycin (10,000 units/mL penicillin and 10,000 µg/mL streptomycin), and 0.25% trypsin-EDTA were purchased from Gibco, Thermo Fisher Scientific, Inc. (USA). JC-1 dye was obtained from Invitrogen, Thermo Fisher Scientific, Inc. (USA). Nar, APAP, dorsomorphin (Dor), and brusatol (Bru) were obtained from MedChemExpress (USA) and dissolved in dimethyl sulfoxide (DMSO). A malondialdehyde (MDA) assay kit (TBA method), superoxide dismutase (SOD) assay kit (WST-1 method), reduced GSH assay kit, and glutamate dehydrogenase (GDH) test kit were obtained from Nanjing Jiancheng Bioengineering Institute (China). A reactive oxygen species (ROS) assay kit was obtained from Beyotime (China). A CCK8 Cell Proliferation and Cytotoxicity Assay Kit was obtained from Solarbio (China).
Antibodies specific for β-actin were purchased from Proteintech Group, Inc. (China). Antibodies specific for mitofusin protein 1 (Mfn1), optic atrophy type 1 (Opa1), phospho (p)-dynamin-related protein 1 (Drp1) (Ser616), total Drp1, AMPK, p-AMPK (Thr172), Nrf2, and GAPDH were purchased from Cell Signaling Technology (USA).

EBTr cells were cultured in DMEM basic medium supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin (J180014, HyClone, Logan, UT, USA) in 37℃ and 5% CO₂. In all experiments, cells were allowed to acclimate for 24 h before any treatments
Cell viability was measured by a CCK8 assay by following the manufacturer’s instructions. In a 96-well plate, 5 × 10³ cells were inoculated for 24 h, after which, different concentrations of LPS (1, 2, 4, 8, or 16 μg/mL) and NAC (0, 0.25, 0.5, 1, 5, 10 mmol/L) were added to the culture medium for 24 h. Then, the CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (Solarbio Life Sciences, Beijing, China) was used to access the survival rate of EBTr cells according to the manufacturer’s instructions.

The biochemical reagent Nevadensin (#3807) was purchased from Nature Standard (Shanghai, China). MG132 (#HY-13,259) and Sorafenib (#HY-10,201) were purchased from MedChem Express. The following antibodies were Used: YAP (#14074T), phospho-YAP (#13619T), MST1(#3682T), phospho-MST1/2 (49,332 S), LATS1 (#3477T), phospho-LATS1 (#8654S), Merlin (#12888T), phospho-Merlin ( #13281S), Cleaved Caspase-3 (#9611S), Cleaved Caspase-9 (#20750S), Cleaved PARP (#5625S), CDK6 (#13,331), Anti-rabbit IgG (#7074), Anti-mouse IgG (#7076) were purchased from Cell Signaling Technology ; CDK4 (#11026-1-AP), Cylcin B1 (#28603-1-AP) were purchased from proteintech; β-actin ( #TA-09 ) was purchased from ZSGB-Bio; DNA Content Quantitation Assay (Cell Cycle) (#CA1510), Hoechst 33,258 (IH0060), CCK-8 Cell Proliferation and Cytotoxicity Assay Kit was (CA1210) purchased from Solarbio. PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) purchased from TaKaRa. FastStart Universal SYBR Green Master (ROX) (#4,710,436,001) purchased from Roche. Cell-Light EdU Apollo 488 in Vitro Kit (100T) (#C10310-1) purchased from RiboBio.

The analytical method was modified according to previous studies [89 (link)]. The effects of the samples on the viability of RAW 264.7 macrophages were measured using the CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (CA1210; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). After overnight culturing in a 96-well plate (2 × 10⁴ cells/well), the cells were treated with various MLF concentrations (0, 5, 50, 150, and 250 μg/mL), which were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) for 12, 24, and 48 h. According to the manufacturer’s instructions, a 10 μL CCK-8 solution was added to each well and incubated at 37 °C for 3 h. The absorbance was measured by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. Each sample had three replicates.

The cell viability was evaluated by CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (CA1210, Solarbio, Beijing, China). HepG2 cells were seeded in 96-well culture plates (5 × 10⁴ cells per well) and were maintained at 37 °C in a humidified atmosphere of 5% CO₂ until to 80% confluence. Cells were then treated according to the purpose of experiment, with six replicates for each treatment. After treatment, the cells were washed with PBS. 10 μL CCK-8 and 100 μL DMEM were then added to each well and incubated for 2 h at 37 °C. The absorbance of each well at 450 nm was read on a Microplate Reader.