Multifunctional microplate reader

MTT assay was used to determine cell viability in order to verify the anti-tumor effect of XJR in vitro. Briefly, colorectal adenocarcinoma DLD-1 cells were seeded in 96-well plates at 1×10⁴/pore. After incubation for overnight at 37°C in 5% CO₂ to allow cell attachment, different concentrations of drug serum were added to the wells and incubated for 48 h. After treatments, the cells were further incubated MTT (Sigma St. Louis, MO, USA) for 4 h. Thereafter, 150 µL/well of DMSO (Sigma St. Louis, MO, USA) was added to the plates. The optical density (OD) at a wavelength of 492 nm was measured using a multifunctional microplate reader (Thermo Scientific., Waltham, MA, USA).

Cells were seeded into 96-well plates (Jet Bio-Filtration, China) in triplicate at a density of 1000 cells per well. Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay (KeyGEN BioTECH) according to the manufacturer’s instructions. The optical density (OD) at 450 nm was measured at the indicated time points using a multifunctional microplate reader (Thermo Fisher Scientific, USA). Cell growth curves were generated using GraphPad Prism software (GraphPad Software, USA).

The activities of caspase-3, caspase-7, caspase-8, and caspase-9 were measured using a Caspase-Glo assay kit (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. The cells were seeded into 12-well plates and were counted with a blood cell counting plate. From each sample, approximately 20,000 cells were added to 100 µL of Caspase-Glo reagent in a 96-well white-walled plate and incubated for 30 min. Luciferase activity was detected using a multifunctional microplate reader (Thermo Scientific, Massachusetts, USA), and the fold increase in protease activity was determined by comparing the luciferase activity of the infected cells with that of mock-infected cells. In the caspase inhibitor assays, caspase inhibitors were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C before use. Cells were exposed to caspase inhibitors (i.e., 20 mM Z-DEVD-FMK, Z-IETD-FMK, Z-LEHD-FMK, and Q-VD-Oph) for 2 h prior to DPV infection.

The cytotoxicity of the films was estimated from the viability of L929 fibroblasts (KAC Co., Ltd., Kyoto, Japan). L929 fibroblasts were cultured at 37 °C in a humidified atmosphere of 5% CO₂ in air, in an eagle’s minimum essential medium (E-MEM, MP Biomedicals, Santa Ana, CA, USA) with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA), 100 U·mL⁻¹ penicillin and streptomycin solution (Gibco, Waltham, MA, USA) and 2.5 μg·mL⁻¹ fungizone (Gibco, Waltham, MA, USA). After culturing for 3 days (Cells covered 80% of plate), the sterilized film was introduced with an insert (filter pore size 8.0 μm) and cultured for 24 h at 37 °C in a humidified atmosphere of 5% CO₂ in air. The cells with only insert is denoted control. The seeded specimens were then incubated with 5 mg·mL⁻¹ 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrasodium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 4 h. The medium was decanted and then the obtained formazan crystals were dissolved with dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and the absorbance was measured at 560 nm with a multifunctional microplate reader (Thermo Scientific, Waltham, MA, USA). The cell viability was determined using Equation (3).