2.6.1. Cells Metabolic Activity

To assess cells proliferation ability, a colorimetric assay (ATCC, Manassas, VA, USA) was used based on the evaluation of mitochondrial dehydrogenase activity to transform water-soluble tetrazole salt (2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolo-5-carboxanilide-XTT) to a soluble formazan product. Three samples were analyzed for each tested material. They were placed individually in 12-well plates (Corning costar) with 2 mL of culture medium. The seeded density in case of each cell line was 6 × 10⁴ cells/cm². Plates were incubated for 48 h at 37 °C in an atmosphere containing 5% CO₂. After this time, medium was removed from each well and all adhered cells (both from the samples and walls of the container) were detached using trypsin. The XTT test was carried out with 100 μL of suspension containing approximately 5 × 10³ of previously obtained cells in culture medium and 0.5 μL of XTT reaction mixture. For the sake of control preparation, the procedure was repeated but without samples in the well). After 4 h of incubation, the absorbance of the mixture was measured at 450 nm and 620 nm. For this purpose, a microplate spectrophotometer (Multiskan GO, Thermo Scientific, Waltham, MA, USA) was used. Cell viability was determined using the following equation:

where: V-cell viability expressed in % of control; A-specific absorbance of the tested mixture; AC-specific absorbance of the control mixture not containing sample.