Cell proliferation and cytotoxicity assay method for CCK8

Posted by beauty33 on November 27th, 2019

What is cytotoxicity?

Cytotoxicity is the process by which a chemical substance (drug) acts on the basic structural  or physiological processes of a cell, such as the cell membrane or cytoskeletal structure, and is also the metabolic process of the cell, the synthesis, degradation or release of cellular components or products, ion regulation, and cell division, as well as adverse reactions caused by cell survival, proliferation or functional disorders. Specific substance cytotoxicity tests, such as drug screening, are sometimes required.

What are the methods for detecting cytotoxicity?

The cytotoxicity test is mainly based on the change of cell membrane permeability, and the following methods are commonly used:

MTT, XTT method: Using the activity of the enzyme inside the mitochondria, the specific tetrazolium salt can be transformed and then detected by a microplate reader;

The principle of detection is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan  and deposit in cells, while dead cells do not. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its light absorption value is measured by a microplate reader at a wavelength of 490 nm (written at 570 nm in the English manual). The amount formed of MTT crystallizes is proportional to the number of cells within a certain number of cells. Based on the measured absorbance value (OD value), the number of living cells is determined. The larger the OD value, the stronger the cell activity (if the drug toxicity is measured, the drug is less toxic).

LDH method: Cytotoxicity is detected by the enzyme activity of LDH in the cell culture supernatant;

Principle of LDH cytotoxicity assay: LDH (lactate dehydrogenase) is a stable protein that is present in the cytoplasm of normal cells. Once the cell membrane is damaged, LDH is released outside the cell. LDH catalyzes lactic acid to form pyruvate and reacts with INT (tetrazolium salts) to form purple crystals, which can be detected by 500nm enzyme labeling instrument. The degree of cell damage can be judged by detecting the activity of LDH in the cell culture supernatant.

Other enzyme methods: Such as detecting the activity of alkaline phosphatase, and acid phosphatase in the supernatant.

Classification and application of cytotoxicity

Cytotoxicity is an adverse reactions caused by the action of a chemical substance (drug) on the cell’s basic structure or physiological processes, such as the cell membrane or cytoskeleton structure, the metabolic process of the cell, the synthesis, degradation or release of cellular components or products, ion regulation, and cell division, causing disturbances in cell survival, proliferation or function. According to the mechanism of action, there are three types:

1. Basic cytotoxicity involving alteration of one or more of the above structures or functions, acting on all types of cells;

2. Selection of cytotoxicity, present in certain differentiated cells, mainly through biotransformation of chemical substances, combined with special receptors or special intake mechanisms;

3. Cell special function toxicity, slight damage to cell structure and function, but the damage to the whole body is very serious. Similar toxic effects can be achieved by the interaction, release, binding and degradation of cytokines, hormones and transmitters that affect cell-to-cell communication or special transport processes. Toxicity may also result from the interference of chemicals with extracellular processes, and any non-animal testing system should consider a variety of factors. In 1983, Ekwall proposed the concept of "basic cell function", that is, the toxicity of most chemicals is a non-specific damage to cell function, but it can cause specific changes in organ function and even death.

Studies have shown that there is a good correlation between the in vitro cytotoxicity of chemicals and its death rate of animals and its plasma concentration of human death. The injury and death caused by chemicals can eventually show changes at the cell level, which speculates that the in vitro cytotoxicity can predict the acute toxicity in vivo. More than 50 years have passed since early research, and in vitro systems that predict acute toxicity in vivo have been developed. The quantitative study between in vitro cytotoxicity and acute toxicity is mainly to analyze the in vitro cytotoxicity IC50 value and the acute toxicity LD50 value of various chemicals in the RC database, and to obtain the RC prediction model for the prediction of acute toxicity LD50 value. Using cytotoxicology to compare the prediction ability  of various endpoints, different tissues and species, it was found that rodent cell lines had good prediction ability to rodent acute toxicity,  and human cell lines have good predictive ability for acute toxicity in humans. BALB/c3T3 cells and human normal keratinocytes (NHK) have good stability and predictive ability in validation experiments, so it is recommended as a common cell line for cytotoxicity analysis. Other cell lines and detection endpoints can also be used. In vitro methods is helpful to predict systemic and local effectscasued by acute exposure to chemicals and to assess the in vivo toxicity concentrations. Therefore, the in vitro cytotoxicity analysis is performed before the acute toxicity test, and then the LD50 value prediction is performed according to the RC prediction model, and the most suitable starting dose for acute toxicity in vivo is selected to reduce the use of the experimental animals.

CCK8 is a fast, highly sensitive detection reagent based on WST-8 that is widely used for cell proliferation and whitening toxicity, often including cell assay services. The method can be used for drug screening, cell proliferation assay, cytotox assay and tumor susceptibility test, and detection of biological factor activity. Some researchers used the CCK8 method to study the effects of different concentrations of red raspberry extract on the proliferation of hepatoma cells.

The basic principle of CCK8 reagent detection:

CCK8 reagent contains WST-8, which is reduced to high water solubility by dehydrogenase in cell mitochondria under the action of electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS). The amount of formazan produced is proportional to the number of living cells. The absorbance of the light at 450 nm is measured by an enzyme-linked immunosorbent assay, which indirectly reflects the number of viable cells. The method has been widely used for the detection of activity of some biologically active factors, large-scale anti-tumor drug screening, cell proliferation test, cytotoxicity test and drug sensitivity test.

Compared with the ordinary MTT method, the CCK-8 method has remarkable features:

★High sensitivity, reliable data and good reproducibility

★Easy to operate, saving time and effort

★Water-soluble, no need to change liquid, especially suitable for suspension cells

★No radioisotope and organic solvents, low cytotoxicity

★It is a bottle of solution, which needs to be prefabricated, ready to use

★Suitable for high-throughput drug screening

CCK-8 use: drug screening, cell proliferation assay, cytotoxicity assay, and tumor susceptibility test.

Cytotoxicity test quality evaluation indicators

The indicators for quality evaluation of cytotoxicity are initially defined as:

1. OD average: Reflects the average level of OD values between the complex wells. The larger the OD value, the more the number of cells.

2. Standard deviation between OD values of duplicate wells: reflecting the dispersion between OD values of each complex well. The larger the standard deviation is, the larger the non-parallel between the duplicated holes is, and the larger the operational error is.

3. Coefficient of variation between OD values: It is also an indicator reflecting the dispersion of OD values between duplicate wells.

4. Survival rate: Calculating the survival rate of cells under each drug concentration can reflect the cell survival. The larger the value, the smaller the cytotoxic effect of the drug.

5. Standard deviation of survival rate: This value reflects that, as each individual well is used as a separate experiment, the flat batch of duplicate wells can be regarded as the distribution of survival rate in several batches of experiments. The larger the index, the less parallel between the re-openings.

6. Coefficient of variation of survival rate: This index reflects the dispersion of survival rate between each re-pore. The larger the value, the greater the non-parallelism between the cells of each column.

7. Half-induced cytotoxic concentration: Refers to the concentration required to produce toxic effects on half of the cells. This indicator can be used to reflect normal work and to evaluate the toxicity of unknown drugs to cells.