Cytotoxicity testing

Evaluation of possible cytotoxicity of various molecules including drug candidates in cell cultures in vitro constitutes first excellent step in toxicological studies that are necessary for flawless determining of drug activity and pharmacological safety. In case of toxicological research in cell cultures in vitro examined molecules, their precursors or metabolites are added to culture media in aim to investigate drug activity on cellular and even molecular level. Applying of cell culture models in toxicological studies has many advantages such as: fast and easy-to-use methods enabling determination of cellular and molecular processes, result reproducibility, requirement of very small quantities of investigated substance, opportunity to search the effect of investigated substance on human cell cultures. Image

BioCentrum offers a broad spectrum of in vitro cell culture preclinical toxicology studies that are performed in compliance with Principles of Good Laboratory Practice (GLP) on adequate selected cellular models. Cytotoxicity research applied in BioCentrum`s Cell Labolatory is depending on necessities executed in both primary cell cultures and cell lines.

MTT and MTS Assays
Both are colorimetric methods for determining the number of viable cells based on mitochondrial dehydrogenase activity measurement. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) tetrazolium compounds are bioreduced by dehydrogenase inside living cells to colored formazan. The quantity of formazan that is directly proportional to the number of living cells in culture is measured spectrophotometically. Bioreduction of MTT compound gives a water-insoluble product that has to be dissolved in organic solvents prior to measurement. MTS assay in turn constitutes improved version of MTT cytotoxicity assay in which formazan product, that is soluble in water is generated in the presence phenazine methosulfate (PMS). Assays are performed by adding a small amount of reagent directly to culture wells, incubating 1-4 hours and then recording the absorbance at 490nm with professional multi-mode 96-well plate reader.

LDH Assay

Similarly to MTT and MTS Assays LDH assay is also a colorimetric test based on production of colored product as a result of enzymatic reaction. LDH assay quantitatively measures lactate dehydrogenase (LDH), intracellular cytosolic enzyme that is released upon cell death and lysis. LDH in culture supernatants is estimated on basis of enzymatic conversion of tetrazolium salt into a red formazan product. The quantity of produced formazan is proportional to the number of lysed (dead) cells. Absorbance measurement data are collected using professional multi-mode 96-well plate reader.

BrdU Assay
Quantitaive estimation of DNA synthesis in cell cultures is now a standard procedure in many laboratories. BrdU assay is a next routine test exploited in cytotoxicity and cell proliferation research in vivo. It is a colorimetric immunoassay that enables quantitative measurement of DNA synthesis and thus cell proliferation. Bromodeoxyuridine (BrdU) is a thymidine analog incorporated into only the DNA of proliferating cells during S phase of cell division process. BrdU incorporation is measured by incubation with specific enzyme-labeled antibody followed by enzymatic reaction with chromogenic substrate. Positive result obtained in BrdU assay renders about cell viability and functionality. Nowadays, BrdU assay is commonly used in research areas such as detection and quantification of cell proliferation induced by growth factors and cytokines, determination of inhibitory or stimulatory of new chemical entities (NCEs) on cell proliferation, lymphocyte immunoreactivity upon mitogen and antigen stimulation, sensitivity of tumor cells to various cytostatic drugs.