Mtt reagent solution

The cytotoxic activity of PldA-GFP IBs was determined by MTT method. After incubation of mouse neuroblastoma Neuro-2a cells with IBs in 96-well microplate for 24 h at 37 °C and 5% CO₂, the supernatant was replaced with pure medium. Then, 5 mg/mL MTT reagent solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and microplate was incubated for additional 4 h, after which SDS-HCl solution (1 g SDS/10 mL dH₂O/17 μL 6 N HCl) was added and incubated at 37 °C for 4–18 h. Absorption was measured at a wavelength of 570 nm using a Multiskan FC spectrophotometer (Thermo Scientific, Canada). The cytotoxic activity of IBs was expressed as the concentration of EC₅₀ at which the metabolic activity of cells is inhibited by 50%.

The MTT assay was used to test the sensitivity of the cells to vemurafenib or etoposide, and was performed as previously described [42 (link)]. Briefly, cells were seeded into 96-well plates and, after 24 h, cells were treated with the indicated doses. After 72 h of treatment, MTT reagent solution (Sigma-Aldrich/Merck) was added into each well and the plate was kept at 37 °C for 3 h. Then, formazan crystals were solubilized with the addition of DMSO, and absorbance was measured at 570 nm with the Infinite M200 (Tecan, Männedorf, Switzerland) or the Multiskan MS (Labsystems) multi-plate readers.

The HaCaT cell line was subcultured in DMEM medium supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin, and incubated in the presence of 5% CO₂ and 95% humidity at 37 °C temperature. A cell suspension of 5 × 10³ cells in 100 µL of DMEM medium was seeded into each well of the 96-well plate and incubated overnight inside an incubator. These wells were treated with 100 µL of DMEM medium containing tea and coffee extracts in such a way that the concentration of tea or coffee extracts was 0.025 mg/mL, 0.05 mg/mL, 0.075 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, and 2 mg/mL inside different wells. The cells inside the control well were not exposed to any extract but only had the DMEM medium. After adding extracts, plates were incubated for 72 h, followed by the addition of MTT reagent solution (20 µL, 5 mg/mL, Sigma Aldrich, USA) to each well and incubated again for 3 h. The excess medium was aspirated, and 100 µL dimethyl sulfoxide (DMSO) was added to each well. The optical density measurements (OD) were performed using a microplate reader (SPARK™ 10M, Tecan, Männedorf, Switzerland) at 570 nm. Relative cell viability was derived from the following Equation (1).
$$\mathrm{Cell} \mathrm{viability} \left(\%\right)=\left(\frac{\mathrm{OD} \mathrm{of} \mathrm{treated} \mathrm{cells}}{\mathrm{OD} \mathrm{of} \mathrm{media} \mathrm{only} \mathrm{controls}}\right)\times 100$$