OPTIMIZATION OF SEVERAL MTT ASSAY PARAMETERS TO EVALUATE THE METABOLIC ACTIVITY OF THE BHK-21 AND SHEEP KIDNEY CELLS

Abstract

The purpose of the work was to define the effect of the amount of formazan on the incubation time of transplanted cell cultures in the MMT assay. Transplanted cultures of Syrian hamster kidney (BHK-21) and sheep kidney (NV-2) cells were cultured in DMEM medium supplemented with 10% fetal calf serum (FBS), Pen Strep antibiotic (Gibco), and Amphotericin B (Gibco) in 96-well tablets in concentrations of 35×10³ and 25×10³ cells/well in 100 μl at 5% CO₂ at a temperature of 37 oC for 24 hours. After that, 100 μl of MTT solution at a concentration of 0.5 mg/ml was added to each well and incubated for 3 or 4 hours. Subsequently, the supernatant was removed and 100 μl of DMSO was added to each well, and the optical density of the contents of the wells was determined after 10, 20, and 30 min of cell incubation at a wavelength of 595 nm. A direct dependence of the amount of formazan formed on the incubation period of NV-2 and BHK-21 cells with the MTT-tetrazolium salt reagent was established. A comparative analysis of the corresponding optical density (OD) values between HB-2 and BHK-21 cell lines indicates a linear dependence of the amount of formazan formed not only on the seeding concentration, but also on the type of cultured cells. Thus, under the conditions of introduction of NV-2 cell suspension at a concentration of 35×103 cells/well in 100 μl of nutrient medium into 96-well plates, after 24 h of cultivation and 4 h of incubation of cells with the MTT reagent (in total, 28 h after seeding), it was established higher OG values compared to the corresponding values of VNK-21 cells that were planted at a higher concentration (40×103 cells/well in 100 μl of nutrient medium). A comparative analysis of the obtained OG values under the conditions of DMSO action on formazan crystals for 10, 20 and 30 min indicated the absence of a significant difference. When planning experiments based on the study of the metabolic activity and viability of NV-2 and BHK-21 cells, the MTT assay should be performed guided by the specified parameters, which will avoid obtaining false results and forming biased conclusions during further experimental in vitro studies.