Infinite 200

The in vitro cytotoxic effect of coumarins 5, 9–12, 15 was evaluated in cancer HeLa cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay³⁵ (link). Cancer cells were seeded in 96-well plates (density of 3 × 10⁴ cells/mL) in 100 μL of medium and cultured for 48 h (80% of cell confluence). Cells were subsequently incubated for 48 h with various concentrations (0.1–100 μM, dissolved in DMSO) of coumarins in culture medium (treated cells). Treated cells were compared for viability to untreated cells (control cells) and vehicle-treated cells (incubated for 48 h with an equivalent volume of DMSO; the maximal final concentration was 1%). After the cell medium removing and washing, cells were subjected to the MTT test³⁵ (link). After incubation (3 h), colour development was measured at 570 nm with an Infinite 200 auto microplate reader (Infinite 200, Tecan, Austria); the absorbance is proportional to the number of viable cells. Two independent experiments were performed. The results were calculated as the percentage of cell viability in comparison with non-treated control cells and expressed as IC₅₀ value (the concentration of compound that reduces the cell viability to 50%).

ROS level. Isolation of mitochondria from brain tissues was performed as described previously [26 (link)]. The ROS production in mitochondria was determined using a respiration buffer (125 mM potassium chloride, 20 mM HEPES, 2.5 mM malate, 2 mM potassium phosphate, 5 mM pyruvate, 1 mM magnesium chloride and 500 μM EGTA, pH 7.0). A mitochondrial extraction was incubated with 25 μM DCF-DA in respiration buffer at room temperature for 20 min. Fluorescence was measured at a wavelength of 530 nm (excitation) and 590 nm (emission)(Infinite 200, Tecan Co., USA).
Mitochondrial membrane potential. mitochondrial membrane potential was conducted using the isolated mitochondria with a 200 μM JC-1. The assay buffer contained mitochondrial isolation buffer with 5 mM pyruvate and 5 mM malate. The assay buffer and mitochondrial extraction were added to the wells of a 96-well black plate, followed by the addition of 1 μM JC-1 and mixed gently. The microplate was covered with aluminum foil and left at room temperature for 20 min. Fluorescence (excitation wavelength: 530 nm, emission wavelength: 590 nm) was then measured (Infinite 200, Tecan Co.).
ATP Content. The previously preprocessed sample was centrifuged at 13,000 ×g for 10 min. The ATP level was measured using a commercial kit (Promega, USA) with a luminescence meter (GloMax-Multi, Promega).

The level of hydrogen peroxide in a mitochondrial suspension was measured as described previously [51 (link)] in standard medium supplemented with 5 mM glutamate, 5 mM malate, 20 µM Amplex Red, horseradish peroxidase (HRP) (3 U/mL), 10 µM EGTA, and, where indicated, 100 µM Ca²⁺, 5 µM menadione, and 2 µM antimycin A. Resorufin accumulation was traced using a plate fluorimeter (Tecan Infinite 200) in 96-well plates at excitation and emission wavelengths of 530 and 595 nm. For the quantitative assessment of hydrogen peroxide, fluorescence was calibrated by an excess of hydrogen peroxide at the end of measurements. In order to avoid light-induced resorufin formation, the fluorescence was measured one or two times a minute.
The rate of SA production was assessed using the highly sensitive chemiluminescent probe 3.7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazine-3-one (MCLA) [52 (link)]. The kinetics of MCLA-derived chemiluminescence (MDCL) was recorded using a plate reader (Tecan Infinite 200) in accordance with a specified protocol [53 (link)]. Some samples contained superoxide dismutase (100 U/mL) for the calibration of luminescent response. Other experimental details are given in figure legend.