M1020

SH-SY5Y, SK-N-AS, and HT-22 cells were cultured in 96-well plates with a density of 1 × 10⁴/well. Different concentrations (0, 12.5, 25, and 50 μM) of luteoloside were used to treat the SH-SY5Y and SK-N-AS cells for 24 or 48 h, while HT-22 was treated with 0, 12.5, 25, 50, and 100 μM of luteoloside. An amount of 20 µL of MTT solution (M1020, Solarbio, Beijing, China) was added to incubate cells in each well for 4 h; then DMSO (D8370, Solarbio, Beijing, China) was used to stop the reaction. Cell proliferation rates were measured using a spectrophotometer (BioTek, Winooski, VT, USA) set at 490 nm.

SH-SY5Y cells (Cat No. CL-0208) were kindly provided by Procell Life Science & Technology and BV2 (Cat No. 1101MOU-PUMC000063) were obtained from the Chinese Academy of Sciences and maintained in DMEM-F12 medium supplemented with 10% heat inactivated fetal bovine serum, penicillin-streptomycin and L-glutamine. This cell line is not listed as a commonly misidentified cell line by the International Cell Line Authentication Committee. A maximum of four cell passages was used. Cytotoxicity induced by MPP⁺ was measured by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method (m1020, Solarbio, Beijing, China). Absorbance was measured with a microplate photometer (Thermo Fisher Scientific, United States) at a wavelength of 490 nm with a reference wavelength of 630 nm.

Cells were seeded into a 96-well plate and routinely incubated. Then, cells were treated with indicated concentrations of SA with or without CCl₄ for 24 h. The cell viability was determined using an MTT assay (M1020, Solarbio Science & Technology Co., Ltd., Beijing, China). The detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT to the water-insoluble blue–violet crystal formazan and deposit it in the cells, while the dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in cells, and the light absorption value can indirectly reflect the number of living cells. The absorbance at a wavelength of 570 nm was quantified using the Emax Precision Microplate Reader.

The A549, H1299, and H460 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. The effect of Cyanidin-3-O-glucoside chloride (C3G) (MB6905, Dalian Meilun Biotechnology Co., Ltd.) on the growth and proliferation of A549, H1299, and H460 cells was measured using the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method and repeated three times. A549, H1299, and H460 cells were seeded in 96-well plates at a density of 10⁴ cells/well and 100 μL/well with RPMI-1640 medium containing 10% FBS. After seeding for 24 h, the medium was removed and replaced with 100 μL of fresh medium containing serial concentrations of C3G (25, 50, 75, 100, 150, and 200 μM). After 48 h of incubation, the cells were treated with 100 μL of fresh medium containing 10 μL 0.5% MTT solution (M1020, Beijing Solarbio Science & Technology Co., Ltd) and incubated at 37 °C for 4 h. The solution was removed, and about 110 μL of DMSO (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well to dissolve the purple formazan crystals. Absorbance was determined at 490 nm by using a microplate reader (Bio Tek, USA). Results were expressed as the percentage of MTT reduction relative to the absorbance of control cells.