Cell Cytotoxicity assay (MTT, XTT and DLH assays)
Types of Cytotoxicity Tests
MTT Assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)
Method: This assay is based on the enzymatic activity of viable cells that convert a compound called MTT into a colored form known as formazan. The amount of MTT converted to the colored form depends on the number of living cells.
Results: A reduction in color absorbance indicates cytotoxicity, as fewer viable cells are able to convert MTT.
Further Explanation:
The MTT assay is used to assess cell viability or to evaluate the cytotoxic effects of drugs or other agents on cells. By affecting intracellular organelles, it can distinguish between live and dead cells. The activity and proliferation rate of cells may be influenced by hormones, growth factors, cytokines, and mitogens, leading to either an increase or no change. Additionally, some drugs and cytotoxic agents, such as anticancer drugs, may cause necrosis, apoptosis, or a decrease in proliferation and growth rates, or result in cell structure degradation.
The MTT assay is a colorimetric method based on the reduction and fragmentation of yellow tetrazolium crystals by succinate dehydrogenase enzymes, leading to the formation of insoluble purple formazan crystals. Unlike other methods, this approach eliminates washing and cell collection steps, which often result in cell loss and increased error. Consequently, all testing stages from cell culture to result reading with a photometer, as well as interpretation of the test, are performed in a microplate, enhancing the assay's repeatability, accuracy, and sensitivity.
The MTT cytotoxicity assay is one of the conventional methods for studying the cytotoxic effects of substances on cells in vitro. In this method, cells cultivated in the laboratory are treated with substances to evaluate their cytotoxicity. The outcome indicates the viability of cells for each concentration of the substance tested. Although this method is primarily designed for soluble compounds and solutions, it is now also applicable to other solvable compounds in organic solvents and nanoparticles. This method is the most widely used for assessing the toxicity of substances and formulations. This toxicity can be examined on unhealthy cell lines, such as cancer cells, for drug studies, or on healthy cells to ensure the safety of compounds.
The MTT assay is a calorimetric test to evaluate cellular metabolic activity. The NADPH-dependent cellular oxidoreductase enzyme reflects the number of viable cells capable of reducing the tetrazolium compound to insoluble purple formazan. The formazan compound is soluble in dimethyl sulfoxide (DMSO). Since dead cells cannot convert MTT to formazan, the level of formazan produced correlates with the number of living cells. The intensity of the resulting color is measured with an ELISA reader.
Cytotoxicity Test Response:
One of the common and rapid methods for assessing the cytotoxicity of drugs is the MTT method, which is based on the formation of formazan color due to the reduction of MTT (dimethyl thiazolyl diphenyl tetrazolium bromide) or other tetrazolium salts. The cleavage of the tetrazolium ring by mitochondrial enzymes in living cells results in the formation of insoluble purple formazan crystals. The formation of these crystals indicates the activity of respiratory chain enzymes and serves as a measure of cell viability. By measuring absorbance using a spectrophotometer at specific wavelengths, the percentage of remaining viable cells can be determined. This percentage is calculated as follows:
Percentage of viable cells = Average absorbance of control samples/Average absorbance of treated samples*100
In this analysis, it is essential to determine the following parameters before conducting the test:
Treatment time: Generally at 24, 48, and 72 hours
Cell line studied: Normal or cancerous
Number of cells plated in each well of the plate
Sample preparation and sterilization method
XTT Assay (Sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium-bis (4-methoxy-6-nitro) benzene sulfonic acid)
Method: Similar to the MTT assay, XTT changes color. Viable cells convert it to a colored form, with the extent of conversion depending on the number of living cells.
Results: The amount of color produced is directly related to the number of viable cells, and a reduction indicates cytotoxicity.
LDH Assay (Lactate Dehydrogenase)
Method: This assay measures the activity of the LDH enzyme in the cell culture medium. LDH is typically found within cells and spills into the culture medium upon cell membrane damage.
Results: An increase in LDH levels in the culture medium indicates cell damage and cytotoxicity.
Trypan Blue Exclusion Assay
Method: In this assay, cells are mixed with Trypan Blue dye, which only stains dead cells. Living cells remain unstained.
Results: Counting the stained (dead) cells relative to the total number of cells is used to assess cytotoxicity.
Comet Assay
Method: This assay is used to evaluate DNA damage in cells. Cells are processed under specific conditions and then assessed for DNA damage using electrophoresis.
Results: DNA damage patterns (resembling comet tails) in cells indicate cytotoxicity and genetic damage.
CellTiter-Glo Assay
Method: This assay is designed to measure ATP (adenosine triphosphate) production, which occurs only in living cells. The amount of ATP measured correlates with the number of viable cells.
Results: A decrease in ATP levels indicates a reduction in the number of viable cells and cytotoxicity.
Steps for Conducting Cytotoxicity Tests
Sample Preparation:
Prepare a sample of the substance or compound to be tested and dilute it to various concentrations.
Cell Culture:
Suitable cells for the assay are cultured and transferred to an appropriate culture medium.
Exposure to Substance:
Cells are exposed to the test substance for a predetermined duration.
Measurement:
After incubation, various cytotoxicity tests are performed to assess the number of viable cells and the extent of damage.
Data Analysis:
The data obtained from the tests are analyzed to determine whether the test substance exhibits cytotoxicity.
Reporting:
The results of the assays are documented in a comprehensive report, including statistical analyses and data interpretation.