Multiskan ascent photometric plate reader

The MTT test is widely used for the assessment of cell proliferation and is based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by the mitochondrial dehydrogenases of the living cells, resulting in the formation of formazan crystals that corresponds with the number of cells. The test was performed according to the already used protocol in our laboratory [13 (link)]. The cells were washed with phosphate-buffered saline, and then 100 μL of the MTT solution at a concentration of 1 mg/mL (MTT, Carl Roth, Karlsruhe, Germany) per well was added to the cells. The cells were incubated with an MTT solution for the next three hours, followed by the dissolution of the formed formazan dissolution with 100 µL of 2-propanol per well. The absorbance of the dissolved formazan was measured on a Multiskan Ascent Photometric plate reader (ThermoLab Systems, Helsinki, Finland) at a wavelength of 540 nm with a correction wavelength of 650 nm. The mean absorbance values were calculated for each tested sample, as well as for the controls. The cell viability and proliferation rate were calculated according to the following formula: % cell viability/proliferation = (absorbance value of treated cells/absorbance value of control cells) × 100.

Quantitative detection of IL-6 was determined by IL-6 ELISA kit (solarbio, Beijing, China). The assay was performed in triplicates, following the manufacturer’s instructions. The plate was read by Thermo Labsystems Multiskan Ascent Photometric plate reader for 96 well plates.