Multimode plate reader

Caspase-3/caspase-9 enzyme activity was measured according to the instructions of the detection kits (catalog numbers: G015-1-3 and G018-1-1, Nanjing Jiancheng Bio-Corporation, Jiangsu, China). Absorbance was measured at 405 nm based on caspase-3/caspase-9 and can catalyze the substrate ac-devd-pna/ac-lehd-pna to produce yellow pNA with strong light absorption value at 405 nm. In brief, 10⁴ cells were cultured and collected from each group followed by adding the reaction solution contained in the kit, and OD value was measured at 405 nm with a multimode plate reader (Thermo Fisher Scientific, United States).

The transfected C666-1 and SUNE-1 cells (2 × 10⁴) were seeded into 96-well plates. At 70% confluence, the cells were washed twice, and BrdU (Cat#: 6813, CST, USA) labeling solution was added to the cells. After 6 h of incubation, the cells were washed twice, fixed, and denatured by the fixation/denaturation solution. The BrdU antibody was then added and the cells were incubated for 2 h. Next, the cells were washed twice, and the secondary horse radish peroxidase (HRP)-mouse antibody was used for another 2 h of incubation. Finally, the cells were washed twice and HRP substrate TMB was added to the cells, and the OD₄₅₀ was obtained on a multimode-plate-reader (Thermo Fisher, USA) [20 (link)].

Ten micrograms of ICA (10 μg)-NDs and the same of ICA (50 μg)-NDs, respectively, were dispersed in PBS (1 mL, pH 7.4) using a bath-type sonicator. After the dispersed samples were carefully transferred into a dialysis membrane bag, the membrane bag was soaked into 15 mL of conical tube containing 5 mL of PBS and then oscillated at 100 rpm and 37 °C. At different incubation time points, such as 1, 5, 9, 12 h, 1, 3, 5, 7, 14, 21, and 28 days, the PBS solution was collected and the same volume of fresh PBS solution was changed. The amount of ICA released was determined at 290 nm using a multi-mode plate reader (Thermo Scientific, MA, USA). Each group was n = 4. This experiment was replicated thrice.

U251 and U87 cells were seeded at a density of 1 × 10⁴ cells/well in 96-well plates. After adherence, the cells were treated with different concentrations of CDDP or TMZ for 48 h, followed by the incubation with 10 μL CCK8 solution (Dojindo, Kumamoto, Japan) for 2 h. Furthermore, a different set of cells was treated with 10 μM CDDP and incubated with 10 μL CCK8 solution for 2 h at four time points (0, 24, 48, and 72 h). The OD value at 450 nm was detected using a multimode plate reader (Thermo Fisher Scientific, MA, USA) [20 (link),22 (link)].

Liver-tissue samples were lysed using lysis buffer (1:10 w/v) for 15 min on ice, then centrifuged at 14,000× g for 15 min at 4 °C. The isolated supernatants were used to determine the activities of caspase-3 and caspase-9 (Beyotime, Haimen, China) using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions, respectively. Total HO-1 activity was measured as described previously [55 (link)]. In brief, a 1.2-mL reaction system consists of 0.5 mg protein from liver-tissue homogenate, 2 mM glucose-6-phosphate, 0.2 U glucose-6-phosphate dehydrogenase, 0.8 mM NADP, and 20.0 mM hemin. The reaction system was incubated for 1 h at 37 °C, then its optical density at 464 nm against a baseline absorbance at 530 nm was determined using a multimode plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The values in the different groups were normalized to the control for statistical analysis.

Cell proliferation was determined using a BrdU kit (Cat#: 6813, CST, USA). Transfected MKN45 and AGS cells (5 × 10³) were cultured in transparent 96-well plates. At 80% cell density, cells were washed, denatured, and incubated with BrdU antibody for 2 h at 25°C. Then, the secondary antibody was added to each well and incubated for 1 h at 25°C. Finally, each well was evaluated by measuring the OD at 450 nm on a multimode-plate reader (Thermo, USA) [23 (link)].

Collagen I solution (Sigma-Aldrich) was added to a 96-well plate for cell adhesion detection, and 2 × 10⁴ U251 and U87 cells were cultured into the plate at 37°C for 4 h. Post culture, the medium was discarded, and the cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent (C0009S, Beyotime, Jiangsu, China) for 2 h at 37°C. Then, 100 µL dimethyl sulfoxide was added to each well, and the OD value at 570 nm was detected using a multimode-plate-reader (Thermo Fisher Scientific) [24 (link)].