Dimethyl sulfoxide (dmso)

Formaldehyde (FA) and benzene (Bz) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). All chemicals were dissolved in 100% dimethyl sulfoxide (DMSO; Junsei Chemical Co., Tokyo, Japan).

DEP cell viability and ROS production were evaluated with MTT (Sigma-Aldrich Corp., St. Louis, MO, USA) and 3.3 μM DCF-DA (Thermo Fisher Scientific, Waltham, MA, USA), respectively. For the cell viability determination, after DEP stimulation, a 1 mg/mL MTT solution was added to the cells and they were then incubated at 37 °C for 3 h. The supernatant was removed and the formazan crystals were dissolved in 100 μL dimethyl sulfoxide (DMSO; Junsei Chemical Co., Tokyo, Japan). Absorbances were measured at 570 nm and the background control was measured at 690 nm in a microplate reader (BioTek, Winooski, VT, USA). Cell viabilities were reported as percentages of the optical density values. To determine the ROS levels, the AM were incubated for 30 min at 37 °C in complete RPMI 1640 medium containing 3.3 μM DCF-DA. The DCF-DA intensities in the cells were immediately measured at 495 nm (excitation) and 529 nm (emission) in a microplate reader. The ROS production levels in the cells were represented as percentages of the DCF-DA intensities relative to the cell viabilities in each well. Moreover, the DCF-DA intensities in the live cells were performed using Cytoflex flow cytometry (Beckman coulter, Brea, CA, USA). The control group was defined as 100%.

Cell viability assays were measured using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. In a 96-well plate, HSC-T6 (6 × 10⁵ cells/well) were cultivated in DMEM medium supplemented as described previously (Vogel et al. 2000 (link)). The effect of ASL on cell viabilities was evaluated by treatment with various concentrations (0.01, 0.025, 0.1, 0.25 and 0.5 mg/mL) for 24 h at 37 °C in an atmosphere of 5% CO₂ and 95% humidity. The cells were then incubated with 0.5 mg/mL MTT (Sigma) for 3 h, and the reaction was interrupted by addition of DMSO (JUNSEI, Tokyo, Japan). An ELISA reader was used to obtain the results at 540 nm. The viabilities of the control cells were used as the control values at 100%.

Lung cancer cell viability was measured by an MTT assay according to the manufacturer's protocols. Briefly, A549 lung cancer cells (1650 cells/well/100 μl media) were placed in a 96-well plate, and then incubated overnight at 37°C. The next day, HB1.F3.CE (3350 cells/well/100 μl media) or vehicle (medium) was added to the existing cancer cells in the 96-well plate. We then applied different concentrations of CPT-11 (Sigma-Aldrich Co. St. Louis, MO, USA) as a prodrug (0.1, 0.2, 0.3, 0.5, 1.0, and 10.0 mmol/l). After four days, 10 μl of 3-(4-,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide (MTT; Sigma-Aldrich Co.) reagent was added to each well, and insoluble formazan crystal was dissolved in dimethyl sulfoxide (DMSO; Junsei Chemical, Tokyo, Japan), then incubated at 37°C for four hours. The absorbance of reduced MTT was measured at 540 nm using a VERSA man microplate reader (Molecular Devices, Sunnyvale, CA, USA). The percent cell viability was plotted using GraphPad Prism (v5.0; GraphPad Software, San Diego, CA, USA). Each data point indicates the average of triplicate measurements (n = 12).