Cytotoxicity refers to the rate of toxic effects in a living cell. A number of tests are performed to understand this ratio. As a matter of fact, the tests that are evaluated by measuring the proliferation rate and toxic effects in the cell are called cytotoxicity tests.
These tests are collected under the main topic 3.
- Tests with tetrazolium salts
- LDH enzyme release test
- They form cytotoxicity tests including luminescence methods.
Tests carried out with the aid of tetrazolium salts: Tetrazolium salts are compounds that are heterocyclic organic compounds. It has been synthesized and identified with more than 1000 members from the years it was discovered (Altman, 1976). Along with the reduction of the tetrazolium salts by taking electrons, it also helps to transform the formazan into a structure called color change. Color reactions are only found in living cells. Tetrazolium tests are experienced in three stages. In the first stage, the living cells are exposed to a certain amount of toxic substance. In the second step, the toxic substance is removed from each other and tetrazolium compound is added and incubated for 1-4 hours. In the last stages, the color change is measured by spectrophotometric method and determination of the number of live or dead cells occurs.
LDH Enzyme Release Test: Another method used in the analysis of cell viability is the determination of lactate dehydrogenase (LDH) activity released from the damaged or dead cells into the medium. (Decker and Lohmann-Matthes, 1988; Korzeniewski and Callewaert, 1983). Lactate dehydrogenase is known as a cytoplasmic enzyme found in all cells. When the cells are exposed to toxic effects, their plasma membrane integrity is disrupted and the LDH enzyme leaks from the cells and passes to the medium. Thus, the damage assessment is carried out by measuring LDH enzyme activity after exposure.
Luminescence Methods:
Alamar Blue Fluorescence Test and Other Fluorescence Methods: The Alamar blue test was first used by Erb and Ehlers (1950) persons for the determination of bacterial contamination in biological fluids and milk, and later in the study of Ahmed et al. (1950) adapted these tests as a side option to the radioactive [1994 H] thymidine incorporation test. This method is based on the conversion of the compound called alamar blue (resazurin) to the resorufin compound together with the living cells. Resazurine is known as an oxidative blue dye redox, which passes freely through the cell membrane to enter the cell, where it is reduced and transformed into a fluorescent pink resorufin compound. Dead cells cannot reduce resazurine and are not capable of generating a fluorescence signal due to loss of metabolic activity. The resulting signal is detected by the use of fluorometers and the intensity increases as the number of viable cells increases.
ATP Bioluminescence Test: Adenosine triphosphate (ATP) molesters act as energy sources with biological systems and can be found in all metabolically active cells. (Crouch et al., 1993). Following cell death, the ATP synthesis state of the cell disappears, and endogenous ATPases perform rapid degradation of readily ATP. For this reason, intracellular ATP content has been described as the main indicator of cell viability and it is becoming one of the methods of viability determination. ATP bioluminescence test has been defined as the fastest, most sensitive and easiest method among the viability tests performed in multiples (Lomakina et al., 2015; Riss et al., 2006).
Real Time Bioluminescence Test: Most of the viability tests are in the form of an end-point format whose cells have not been destroyed or allowed to be further investigated, making kinetics and real-time analysis of toxic substances impossible. Because toxicity is time and dose dependent, the effects of different dose ranges can only be understood by studying within different times. Similar to the ATP bioluminescence assay, the luciferase enzyme was also used in this assay. However, the luciferase enzyme used in this assay was obtained from a different organism. As the luciferase process, a substance called coelenterazine is used.
You can work with our laboratory EUROLAB for cytotoxicity tests.