Mtt cell proliferation and cytotoxicity assay kit

The MTT Cell Proliferation and Cytotoxicity Assay kit was purchased from Sangon Biotech (Shanghai, PR China), AG490 from Beyotime(Jiangsu, PR China) and IL-6 from eBioscience (eBioscience, CA, USA). Primary antibodies against β-actin and GAPDH were purchased from Santa Cruz (Shanghai, China), antibody targeting GRIM-19 from Abcam (ab3449, Shanghai, PR China), and antibody against p- STAT3 (#4113), total STAT3 (#12640), p- AMPKα (Thr172, #2531), total AMPKα (#2532, p- Akt (Ser473, #4058) and total AKT (#4685) were from Cell Signaling Technology (Danvers, MA, USA). And all the secondary antibodies were purchased from Biosynthesis Biotechnology (Beijing, PRChina); penicillin, streptomycin, DMEM medium, and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NY, USA).

PAC₂, a zebrafish embryonic cell line, was gifted from Dr. Shawn Burgess’ laboratory. Cells were cultured in Leibovitz L15 medium supplemented with 15% fetal bovine serum and antibiotics12 (link). Cell proliferation and cell viability was determined by MTT(3-[4,5-dimethyl thiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay (MTT Cell Proliferation and Cytotoxicity Assay Kit, Sangong Biotech, Shanghai, China), which also revealed the integrity and activity of mitochondria, as described13 . The PAC₂ cells were seeded in 96-well tissue culture plates at a density of 1 × 10⁵ cells/ml. After reaching 80% confluence, the cells were incubated with different concentrations of the starfish tissue extracts (0.8, 4, 20, and 100 μg/ml) for 24 h. DMSO was used as a control. The attached cells were washed twice with phosphate buffer solution (PBS). Twenty μl of MTT stock solution (5 mg/ml in PBS) were added to each well, and the plates were further incubated overnight at 32 °C. One hundred μl of DMSO were added to each well to solubilize the formazan crystals produced by viable cells. After complete dissolution, the plates were agitated for 10 min, and absorbance was detected at 490 nm using a fluorometric ELISA plate reader (Spectramax, Gemini EM). This procedure was performed in triplicate in a parallel manner for each concentration.

MTT assay was used to screen the cytotoxic or protective activity of the water extract compound from NXT. The cell viability was determined with MTT Cell Proliferation and Cytotoxicity Assay Kit (Sangon) according to the manufacturer's instructions. Cells (1 × 10³ cells/well) were seeded in 96-well plates. After the corresponding treatment, cells were washed twice with PBS and then incubated with the MTT solution for 1.5 h at 37°C. Cells were then dissolved in 200 μL DMSO in 96-well plates. The absorbance of the reaction solution at 570 nm was measured with an ELISA-plate reader, Multidetection Microplate Reader Synergy H4 (BioTek).

Cell proliferation ability was estimated through MTT assay using MTT Cell Proliferation and Cytotoxicity Assay Kit (Sangon Biotech). In brief, cells were seeded to a 96‐well plate with a density of ×10⁵ cells/mL. Then, the cells were incubated at 37°C with 5% CO₂. At an interval of one day, 20 μL MTT was supplemented into cell medium and incubated for an additional 4 hours. Then, 150 μL DMSO was added and incubated at dark for 10 minutes to stop reaction. Subsequently, a microplate reader (TECAN) was used to detect the absorbance of the cell medium at 490 nm to estimate cell proliferation. Each test was carried out in triplicate.

The cell proliferation was assessed using a MTT cell proliferation and cytotoxicity assay kit (Sangon Biotech), according to the manufacturer’s instructions. In brief, cell culture medium was removed, and cells were treated with 1:1 diluted MTT solution in fresh serum-free medium for 3 h at 37°C. Following incubation, MTT solvent was added into each well and incubated on an orbital shaker for 15 min. Finally, the samples were read at OD590 nm with a microplate reader.