Mithras 940

Cell viability was evaluated using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as previously described [14 (link)]. Briefly, BJ and B16 cells were seeded in 96-well plates at densities of 10,000 cells/well and 7000 cells/well and further cultured in the medium for 24 h. The next day, the investigated compounds were added at a concentration of 1 to 25 μM for 24 and 48 h. The cells cultivated in medium without the studied samples served as negative controls. After incubation for the desired times, the medium was replaced with MTT solution (1 mg/mL) and incubated for an additional 4 h at 37 °C. Finally, the medium was collected, and DMSO was used to dissolve the insoluble formazan product. A Mithras 940 (Berthold, Bad Wildbad, Germany) plate reader was used to measure the absorbance of the samples at 570 nm. The data were corrected for background, and the percentage of viable cells was obtained using the following equation:
Half-maximal inhibitory concentration (IC₅₀) was determined by fitting the data with a sigmoidal logistic function using Origin 8.1 (Microcal Inc., Northampton, MA, USA) software.

The effect of biohybrids on the cells’ viability was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, as described previously [48 (link)]. First, the cells were seeded in 96 well plates and further cultured for 24 h. Following, the samples were added into the medium for 24 h. As negative control we used cells grown only in medium. After one day, the medium was discarded and 1 mg/mL MTT solution was added to each well and incubated for additional 4 h at 37 °C. Finally, the solution was discarded, and the insoluble formazan product was dissolved in DMSO. Finally, the samples absorbance was recorded using a plate reader Mithras 940 (Berthold, Germany) at 570 nm. All values were corrected for the background by subtracting the blank and cell viability was obtained using the following equation: [(A₅₇₀ of treated cells)/(A₅₇₀ of untreated cells)] × 100%. The sample’s concentration that was able to reduce cell viability by half (IC₅₀) was extracted by fitting the experimental data with a logistical sigmoidal equation in Origin 8.1 software (Microcal Inc., Los Angeles, CA, USA).

To test cellular viability, we used the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay as previously described [51 (link)]. Briefly, L929 and B16 cells were seeded in 96-well plates at a density of 5000 cells/well and grown in the culture medium for 24 h. The next day, the cells were treated with the investigated compounds, added in concentrations of 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, and 25 μg/mL for 24 and 48 h, respectively. The cells cultivated without the tested compounds served as negative controls. After the incubation period, the culture media was removed, and the MTT solution was added at a final concentration of 0.5 mg/mL. This assay is based on transforming the tetrazolium salt into formazan crystals in the metabolically active cells. After 4 h of incubation at 37 °C, the formazan crystals were dissolved using DMSO. The absorbance of the samples was recorded at λ = 570 nm using a plate reader (Mithras 940, Berthold, Bad Wildbad, Germany). The data were corrected for background absorbance, and the viability was calculated using the following formula:
Half-maximal inhibitory concentration (IC₅₀) was determined by fitting the data with a sigmoidal logistic function using Origin 8.1 (Microcal Inc., Northampton, MA, USA) software.