First, the principle of CCK8 detection of cell proliferation / toxicity
Cell Counting Kit-8 (CCK-8) reagents are used for simple and accurate cell proliferation and toxicity analysis. The basic principle is: the reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4 - benzene disulfonate - 2H-tetrazole monosodium salt], which is in the cell under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS) The dehydrogenase is reduced to a highly water-soluble yellow formazan dye. The amount of formazan produced is proportional to the number of living cells. This feature can therefore be used to directly perform cell proliferation and toxicity analysis.
 Uses : drug screening, cell proliferation assay, cytotoxicity assay, tumor susceptibility test
Advantages of CCK8:
Easy to use, eliminating the need to wash cells, no radioisotopes and organic solvents;
CCK-8 method can detect quickly;
The CCK-8 method has high detection sensitivity and can even measure lower cell density;
The repeatability of the CCK-8 method is superior to the MTT method;
The CCK-8 method is less cytotoxic;
The CCK-8 cell activity assay reagent is a bottle of solution that needs to be prefabricated and ready to use.
Disadvantages of CCK8 :
Compared with the MTT method, the prices of CCK8 and XTT are relatively expensive.
The color of the CCK8 reagent is light red, which is close to the color of the medium containing phenol red. If it is not noticed, it is easy to cause leakage or excessive addition.
Compared with previous proliferation/toxicity assay reagents:
Second, CCK8 method for detecting cell proliferation/toxicity
Experiment 1: Cell proliferation analysis
1. Preparation of cell suspension: cell count
2. Inoculate into 96-well plates: According to the number of suitable plating cells, about 100 ul of cell suspension per well, the same sample can be done in 3 replicates.
3. Culture in a 37 ° C incubator: After the cells are inoculated, it takes about 2-4 hours to adhere to the wall. If it is not required to adhere, this step can be omitted.
4. Add 10ul CCK8: Since the amount of CCK8 added per well is relatively small, it may cause errors due to the reagent being stuck on the pore wall. It is recommended to gently tap the culture plate after the reagent is added to help mix. Alternatively, the medium containing 10% CCK8 was directly dispensed and added as a liquid exchange.
5, culture 1-4 hours: different types of cells, the amount of Formazan formed is not the same. If the color is not enough, you can continue to train to confirm the best conditions. In particular, Formazan, which is formed by blood cells, is rare and requires a long color development time (5-6 hours).
6. Determination of absorbance at 450 nm: It is recommended to use dual wavelength measurement, the detection wavelength is 450-490 nm, and the reference wavelength is 600-650 nm.
Experiment 2: Cytotoxicity analysis
1. Preparation of cell suspension: cell count
2. Inoculate into 96-well plates: According to the number of suitable plating cells, about 100 ul of cell suspension per well, the same sample can be done in 3 replicates.
3. Culture in a 37 ° C incubator: After the cells are inoculated, it takes about 2-4 hours to adhere to the wall. If it is not required to adhere, this step can be omitted.
4, adding different concentrations of toxic substances
5, 37 ° C incubator culture: the time of adding toxic substances, depending on the nature of the toxic substances and cell sensitivity, generally depends on the cell cycle to decide, at least for more than one generation.
6. Add 10ul CCK8: Since the amount of CCK8 added per well is relatively small, it may cause errors due to the reagent being stuck on the pore wall. It is recommended to gently tap the culture plate after the reagent is added to help mix.
7, culture 1-4 hours: different cell types, the amount of Formazan formed is not the same. If the color is not enough, you can continue to train to confirm the best conditions. In particular, Formazan, which is formed by blood cells, is rare and requires a long color development time (5-6 hours).
8. Determination of absorbance at 450 nm: It is recommended to use dual wavelength measurement, the detection wavelength is 450-490 nm, and the reference wavelength is 600-650 nm.
Note:
If the OD value is not measured temporarily, 10 μL of 0.1 M HCL solution or 1% w/v SDS solution may be added to each well, and the culture plate is covered and stored at room temperature in the dark. The absorbance does not change within 24 hours.
If the substance to be tested is oxidizing or reducing, replace the medium with fresh medium (removal of the medium and wash the cells twice with medium, then add new medium) to remove the effects of the drug. Of course, in the case where the influence of the drug is relatively small, the medium can be directly subtracted from the blank absorption after the drug is added to the medium.
Third, CCK8 to detect cell proliferation / toxicity considerations
When using standard 96-well plates, the minimum inoculum size of adherent cells is at least 1,000 per well (100 μl of medium). Sensitivity when detecting leukocytes is relatively low, so the recommended inoculum size is not less than 2,500 per well (100 μl medium).
Phenol red and serum do not interfere with the detection of the CCK8 method and can be eliminated by subtracting the absorbance of the background in the blank well.
CCK8 can detect E. coli but not yeast cells. It is necessary to avoid bacterial contamination during each measurement of the cell proliferation experiment so as not to affect the results.
CCK-8 can be stored for at least 6 months at 0-5 ° C and 1 year at -20 ° C in the dark.
When cultured in an incubator, the outermost circle of the plate is most likely to dry and volatilize, increasing the error due to inaccurate volume. Under normal circumstances, only the medium is added to the outermost circle, and it is not used as a measuring hole.
CCK8 was added to the culture medium for a certain period of time, and the absorbance at 450 nm was determined as a blank control. In the dosing experiment, the absorption of the drug should also be considered. CCK8 can be added to the medium to which the drug is added, cultured for a certain period of time, and the absorbance at 450 nm is measured as a blank control.
Metals have an effect on CCK-8 color development: when the final concentration of 1 mM of lead chloride, ferric chloride, copper sulfate will inhibit 5%, 15%, 90% of the color reaction, the sensitivity is degraded. If the final concentration is 10 mM, it will be 100% inhibited.
Suspension cells are generally difficult to stain because of the increased number of cells and prolonged culture time.
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