Advantages and usage of CCK-8 cell proliferation and toxicity detection kit
Pubtime：2024-01-25
View volume：253
Cell Counting Kit-8 (CCK-8 kit) is a rapid and highly sensitive colorimetric detection kit widely used for cell proliferation and cytotoxicity based on WST-8 (chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H tetrazole monosodium salt). The detection principle is that WST-8 can be reduced by some dehydrogenases in the mitochondria in the presence of electron coupling agent 1-Methoxy PMS to generate orange yellow water-soluble formazan (refer to Figure 1). The color intensity of the generated formazan is proportional to cell proliferation and inversely proportional to cytotoxicity. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at a wavelength of 450nm, indirectly reflecting the number of live cells. This product can be used for cell proliferation assay, cytotoxicity detection, drug screening, and cell growth inhibition detection.
Advantages of CCK-8 method
| test method | MTTmethod | XTTmethod | WST-1method | CCK-8method |
| Product characteristics | powder | 2Bottle solution | solution | 1 bottle of solution |
| usage method | Used after preparing the solution | Ready to use, ready to use | Ready to use | Ready to use |
| detection sensitivity | chigh | Very high | Very high | high |
| Testing time | longer | Shorter | Shorter | minimum |
| Detection wavelength | 560-600nm | 420-480nm | 420-480nm | 430-490nm |
| Cytotoxicity | High, disappearance of cell morphology * | Very low, unchanged cell morphology | Very low, unchanged cell morphology | Very low, unchanged cell morphology |
| Reagent stability | commonly | Poor | commonly | very good |
| Batch sample testing | sure | Very suitable | Very suitable | Very suitable |
| Convenience level | commonly | convenient | convenient | Very convenient |
Transportation and storage
Blue ice transportation- Stored at 20 ℃, with a shelf life of 24 months. [Note]: Repeated freeze-thaw cycles can cause an increase in the measured background OD value.
usage method
Taking a 96 well plate as an example, 100 μ L of cell suspension is inoculated into each well of the plate and pre cultured in a cell culture incubator for 24 hours.
Add 10 μ L of the test substance of different concentrations to each well. If only cell activity is measured, steps 2-3 are not required, and the operation can proceed directly from step four.
Incubate the culture plate in the incubator for an appropriate period of time (depending on the drug being tested).
Add 10 μ L of CCK-8 solution to each well (be careful not to generate bubbles, mix gently).
Incubate the culture plate in the incubator for an appropriate period of time (usually 0.5-2 hours).
Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.
matters needing attention
This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields。
Suggest adjusting the appropriate number of inoculated cells and the cultivation time after adding CCK-8 solution. When using a standard 96 well plate, there should be at least 1000 adherent cells per well (100 μ L medium) and at least 2500 white blood cells per well (100 μ L medium). It is recommended to set up several gradient wells with different cell numbers for condition testing before the experiment. The cell culture time and treatment method should be determined according to the experimental situation. After adding CCK-8 solution (with a volume of 10% of the cell culture medium volume per well), incubate in a 37 ℃ cell culture incubator, and measure the absorbance value at 450 nm at different time points (CCK-8 has high sensitivity, and some cells can be measured for the first time after 0.5 hours).
The detection of CCK-8 kit relies on dehydrogenase catalyzed reaction. If there are too many reducing agents (or antioxidants) in the system to be detected, it will cause an increase in background OD value and interfere with the detection results. If there is a reducing agent in the experiment, please check the OD value of the background and try to remove the interference of the reducing agent first. For example, fresh culture medium can be replaced before adding CCK-8 solution to remove the influence of the test drug. When the effect of the test drug is relatively small, it is not necessary to replace the culture medium, and the blank absorption after adding the test drug to the culture medium can be directly deducted.
When conducting drug inhibition experiments, if the drug contains metals such as Pb2+, Fe2+, Cu2+, etc., they will affect the color reaction of CCK-8 Solution, resulting in a decrease in detection sensitivity.
If the cell culture time is too long and the color of the culture medium changes, the cells should be washed and the culture medium should be replaced before adding CCK-8 detection. Phenol red in cell culture medium does not affect the measurement results.
To ensure thorough mixing of the CCK-8 reagent kit culture medium and reduce residual CCK-8 on the pipette, it is recommended to dilute the CCK-8 reagent with the culture medium before adding the sample, and then add the sample after mixing.
If the OD value is not measured temporarily, 0.1 M HCL solution or 1% w/v SDS solution can be added to each well, stored at room temperature in the dark, and detected within 24 hours without affecting the absorbance. The volume of 1% SDS solution added is the same as the volume of CCK-8 solution added.
If the absorbance value is very low, the number of cells can be appropriately increased or the incubation time after adding CCK-8 solution can be extended.
For your safety and health, please wear lab coats and disposable gloves when operating.
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