3.3.2. Cell Viability Assay

## MTT Assay

Stocks of substances were prepared in DMSO at a concentration of 10 mM. All studied compounds were tested in concentrations from 25 μM using twofold dilution. All tested compounds were added to the wells of the plates in a volume of 20 μL diluted in PBS (final DMSO concentration < 1%). A known cytotoxic agent—triterpene glycoside cucumarioside A2-2 from holothurian *Cucumaria japonica*—was used as positive control. Cells (3 <sup>×</sup> 104 cells/well) were incubated with different concentrations of 1,4-NQs in a CO2 incubator for 24 h at 37 ◦C. After incubation, the medium with tested substances was replaced with 100 μL of pure medium. Then, 10 μL of MTT (Sigma-Aldrich, St. Louis, MO, USA) (thiazolyl blue tetrazolium bromide) stock solution (5 mg/mL) was added to each well and the microplate was incubated for 4 h. After that, 100 μL of SDS-HCl solution (1 g SDS/10 mL dH2O/17 μL 6 N HCl) was added to each well followed by incubation for 4–18 h. The absorbance of the converted dye formazan was measured using a Multiskan FC microplate photometer (Thermo Scientific, USA) at a wavelength of 570 nm [32]. The results were presented as percent of control data, and concentration required for 50% inhibition of cell viability (EC50) was calculated. Selectivity index (SI) is defined as the ratio of EC50 for normal cells and EC50 for tumor cell lines (SI = EC50 (normal cells)/EC50 (tumor cells)).

#### Nonspecific Esterase Activity Assay

The test solution (20 μL) and 180 μL of the cell suspension were placed into each well of a 96-well microplate (3.5 <sup>×</sup> 10<sup>4</sup> cells/well). The plates were incubated in a CO2 incubator at 37 ◦C for 24 h. A stock solution of the probe fluorescein diacetate (FDA) (Sigma-Aldrich, St. Louis, MO, USA) in DMSO (1 mg/mL) was prepared. After incubation, cells were washed with PBS, 10 μL of FDA solution (50 μg/mL) was added to each well and the plate was incubated at 37 ◦C for 15 min, and fluorescence was measured with a Fluoroskan Ascent plate reader (Thermo Labsystems, Finland) at λex = 485 nm and λem = 518 nm. All experiments were repeated in triplicate. Cytotoxic activity was expressed as the percent of cell viability.

## Hemolytic Assay

Erythrocytes were used at a concentration that provided an optical density of 1.0 at 700 nm for a non-hemolyzed sample. In addition, 20 μL of a solution of test substances with a fixed concentration was added to a well of a 96-well plate containing 180 μL of the erythrocyte suspension. The erythrocyte suspension was incubated with substances for 1 h at 37 ◦C. After that, the optical density of the obtained solutions was measured at 700 nm and EC50 for hemolytic activity of each compound was calculated.

#### 3.3.3. Antimicrobial Assay
