Cell biochem biophys is a branch of biology that studies the physical aspects of the existence of living nature at all its levels from molecules and cells to the biosphere as a whole. It is a section of modern mathematical physics that studies biological objects as a kind of complex nonlinear physical systems; the science of physical processes occurring in biological systems of different levels of organization, and the impact on various biological objects of various physical factors. Biophysics is designed to identify the relationship between the physical mechanisms that underlie the organization of living objects, and the biological characteristics of their life.
Under the influence of physiologically active substances, cells can undergo changes in morphology, cell growth rate, death time and degree of disintegration, therefore, for each substance, which is a potential pharmacological agent, it is necessary to evaluate the effect on cell survival. There are various methods for determining the cytotoxicity of organic synthesis products, natural compounds, and extracts. These methods can be divided into three groups:
- measurement of mitochondrial activity;
- assessment of the release of lactate dehydrogenase or adenylate kinase due to a violation of the cytoplasmic membrane integrity;
- determination of the ATP amount in cells and the activity of 3/7 kinase as indicators of cell necrosis and apoptosis.
The MTS assay, which was first described by Mosmann for over 30 years ago, is widely used as a screening method for measuring cell survival and is included in most protocols of molecular biology and medicine. It is based on the reaction of the reduction of the yellow tetrazolium salt (MTS) by mitochondrial dehydrogenases (hoechst nuclear stain) of living cells to purple formazan crystals, which are insoluble in the aquatic environment of the cells. Crystals of formazan (MTS-f) are dissolved in DMSO or in a mixture of HCl-isopropanol and determined colorimetrically. This combination kills living cells, that is, the MTS assay is the final study point. In addition, the method of determining cytotoxicity using MTS assay is poorly implemented when using suspension cultures, since it implies the complete removal of the culture medium at the stage of dissolution of formazan crystals. However, the advantages of this method are cost-effectiveness and excellent reproducibility of the results.
Differential Nuclear Staining (DNS) assay adgusted to live-cell imaging for high throughput screening (HTS). It uses two fluorescent DNA intercalators:
- Hoechst 33342;
- Propidium iodide (PI).
This nuclear staining was applied to count the total number of cells. The DNS assay was successfully verified by using well known cytotoxic agents with fast or slow cytotoxic properties. The test was created to be appropriate for HTS with Z’ factors. It ranges from 0.86 to 0.60 for 96 and 384-well formats, respectively. Additionally, the DNS assay was utilized to verify the cytotoxic compounds’ activity. The DNS assay was also applied to produce dose-response curves and to acquire CC50 indicators. The outcomes point out that the DNS assay is trustworthy and fit for primary and secondary screens of compounds that have potential cytotoxic activity.