Cytostatic Effect Evaluation Protocol

Cells were distributed into 96-well tissue culture plates with an initial cell number of 5.0 × 10³ per well. After 24 h incubation at 37 °C, the cells were treated with the compounds in 200 µL final volume containing 1.0 v/v% DMSO and 10% H₂O. The cells were incubated with the compounds at 3.125–25 µM concentration range for 24 h, whereas control cells were treated with serum-free medium only or with DMSO (c = 1.0 v/v%) and H₂O (c = 10.0 v/v%) at 37 °C for 24 h. After incubation, the cells were washed twice with a serum-free medium. To determine the in vitro cytostatic effect, the cells were further cultured up to 72 h in a 10% serum-containing medium. A solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and MTT-solution, (45 µL, 2 mg/mL, final concentration: 0.37 µg/mL) were added to each well. The respiratory chain [67 (link)] and other electron transport systems [68 (link)] reduce MTT, and thereby form non-water-soluble violet formazan crystals within the cell [69 (link)]. The amount of these crystals can be determined spectrophotometrically and serves as an estimate for the number of mitochondria, and hence the number of living cells in the well [70 (link)]. After 3 h of incubation, the cells were centrifuged for 5 min at 1000 g and the supernatant was removed. The obtained formazan crystals were dissolved in DMSO (100 μL) and the optical density (OD) of the samples was measured at λ = 540 and 620 nm, respectively, using ELISA Reader (iEMS Reader, Labsystems, Finland). OD₆₂₀ values were subtracted from OD₅₄₀ values. The percentage of cytostasis was calculated by using the following equation:
Values of OD_treated and OD_control correspond to the optical densities of the treated and the control cells, respectively. In each case, two independent experiments were carried out with four parallel measurements. The 50% inhibitory concentration (IC₅₀) values were determined from the dose–response curves. The curves were defined using Microcal™ Origin 2018 software: cytostasis was plotted as a function of concentration, fitted to a sigmoidal curve, and based on this curve, the half-maximal inhibitory concentration (IC₅₀) value was determined. IC₅₀ represents the concentration of a compound that is required for 50% inhibition in vitro and is expressed in micromolar units.