Methyl thiazolyl tetrazolium (mtt)

Referring to Jin et al. (2019) , Madin-Darby bovine kidney cells (MDBK, Jennio, Guangzhou, China) grown to 80% confluency were digested with 0.25% trypsin digestion solution (BioFroxx, Guangzhou, China). Cells were seeded in a 24-well culture plate (1 × 10⁴ cells/well) and incubated in a cell culture incubator (5% CO_2, 37 °C, 24 h). When the cells in the culture plate grew to 80% confluency or above, morin was added at 31.25, 62.5, 125, 250, 500, and 1000 μM. Blank control and three replicates of each group were set, with continuing incubation at 5% CO₂/37 °C. After 48 h incubation, 10 μL MTT (2,5-diphenyl-2H-tetrazolium bromide) reagent (5 mg/mL, Roche, Beijing, China) was added to each well and then cultured in the cell culture incubator for 4 h. A 100 μL MTT solution was added to dissolve Metzan crystal in each well, followed by shaking on a rotating shaker for 10 min. A multifunctional microplate reader was used to measure the absorbance at 550 nm. The OD₅₅₀ values of the experimental and the blank control groups were determined to judge the cell viability and the toxicity of the inhibitors on the cells. Data were processed using GraphPad Prism7 software, and CC₅₀ values (half cytotoxicity concentration) were calculated. All cytotoxicity tests were repeated three times.

Cells were cultured in a humidified incubator maintained at 37°C and 5% CO₂ environment. Cells were cultured in complete medium (DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin). DoxR cells were generated by incubating parental WT cells with incremental concentrations of doxorubicin ranging from 1 nM to 1 μM over a 6-month period. Treatment began with 1 nM and was increased to the next 10-fold increment after surviving 5 consecutive passages. Cells were considered to be resistant after surviving 5 consecutive passages in 1 μM doxorubicin. Cell viability was determined by the quantitative colorimetric MTT assay according to Boehringer Mannheim (Cat. No. 1465007) as previously described [23 (link)].

The MTT cell viability assay was performed in DL (cancer cell) and peripheral blood mononuclear cells (PBMC) (normal cell) according to the instructions in the kit (MTT, manual from Boehringer Mannheim, Cat. No. 1465 007). After incubation with different concentration (0, 0.01, 0.1, 0.5, 1, 5 and 10 μM) of both compounds for 24 hours, 10 μL of the MTT labeling reagent (0.5 mg mL⁻¹) was added into each well except the empty wells. The microtiter plate was then incubated for four hours in a humidified atmosphere, at 37 °C and 5% CO₂. After that, 100 μL of the DMSO was pipetted into each well. The plate was checked for complete solubilization of the purple formazan crystals under the inverted phase microscope followed by measurements of optical density at the wavelength of 550 nm.^{38,39 (link)} The percentage of cell viability was calculated using the following formula:A dose–response curve (% cell viability versus sample concentration) was plotted and the sample concentration that inhibits 50% of the cell viability (IC₅₀) was determined.

Before cytotoxicity test, each scaffold (6 × 6 × 5.5 mm³) was sterilized in 70% ethanol for 3 h, washed in deionized water, and then irradiated with ultraviolet (UV) for 30 min. Each scaffold was immersed in culture medium (100 mg/mL) consisting of DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/mL penicillin (Thermo Fisher Scientific), and 100 mg/mL streptomycin (Thermo Fisher Scientific) at 37°C for 24 h. The extracts were collected for cytotoxicity test. Phenol (1%) was used as positive control and PCL and DMEM as negative control, respectively.
Mouse fibroblast-like cells (L929 cells; Korean Cell Line Bank, Seoul, Korea) were seeded in 48-well culture plate (5×10⁴ cells/cm²) and cultured for 24 h with each extract. Then a MTT (Roche Applied Science, Indianapolis, IN, USA) assay was carried out to quantify the amount of viable cells. Briefly, MTT labeling reagent was added to each well and the plates were incubated for 4 h at 37°C. Solubilization solution was then added and incubated overnight at 37°C. The OD of each well was measured at 570 nm in a microplate reader (Multiskan EX; Thermo Fisher Scientific).