Structure Search


    Online Support

  •  Customer service

    Location: Thematic focus

Cell proliferation assay - MTT

2019-07-15 来源:亚科官网
Cell proliferation refers to the process in which cells undergo cell division through reactions such as DNA replication under cyclically regulated factors. Proliferation detection generally analyzes the changes in the number of cells in the division, and then reflects the growth state and activity of the cells. Currently, it is widely used in the fields of tumor biology, molecular biology, and pharmacokinetics.
Method for detecting cell proliferation
There are many methods for detecting cell proliferation, and currently there are mainly two methods for detecting cell proliferation ability.
One is a direct method to evaluate the proliferative capacity of cells by directly measuring the number of cells that divide. For example: Ki67 cell proliferation assay; BrdU cell proliferation assay; EdU cell proliferation assay.
The other is an indirect method, a cell viability assay, which evaluates the proliferative capacity of a cell by detecting the number of healthy cells in the sample. Obviously, the cell viability assay does not ultimately prove whether the cells in the test sample are proliferating. For example: MTT method, CCK8 kit method, the following describes the MTT method.
What is MTT?
MTT is a powdered chemical called 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide. Trade name: thiazole blue. It is a yellow color dye, CAS No. : 298-93-1
Principle of MTT
The principle of detection is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT to water-insoluble blue-purple crystalline formamidine and deposit in cells, while dead cells do not. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and the 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 of MTT crystal formation is within a certain cell number range. It is proportional to the number of cells. According to 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 toxicity is smaller).
Experimental steps of MTT
1. Inoculate cells
The culture medium containing 10% fetal calf serum was mixed into a single cell suspension, and 1000-10000 cells per well were seeded into a 96-well plate at a volume of 200 uL per well.
2. culture cells
Same with the general culture conditions, culture for 3~5 days (the culture time can be determined according to the purpose and requirements of the test).
3. color
After 3-5 days of culture, add MTT solution (5 mg/mL with PBS) to each well for 20 uL. Continue incubation for 4 hours, terminate the culture, carefully discard the culture supernatant in the well, and centrifuge the cells for centrifugation. 150 uL of DMSO was added to each well and shaken for 10 minutes to allow the crystals to fully melt.
4. colorimetric
The wavelength of 490 nm was selected, and the light absorption value of each well was measured on an enzyme-linked immunosorbent monitor. The results were recorded, and the cell growth curve was plotted with time as the abscissa and absorbance as the ordinate.
Experiment considerations
1. Select the appropriate cell inoculation concentration.
2. To avoid serum interference: generally choose less than 10% of fetal bovine serum culture medium for testing. After coloring, try to absorb the residual culture solution in the well.
3. Set a blank control: a blank control in which the culture solution was added without parallel to the test. The other test steps are consistent, and the final colorimetric is zeroed with a blank.
The absorbance of the MTT experiment is finally between 0 and 0.7. If it is outside this range, it is not a linear relationship.
IC50 is the half-inhibition rate, which means the concentration of the drug at the inhibition rate of 50%. The drug is diluted to different concentrations, and then the respective inhibition rates are calculated. The drug concentration is plotted on the abscissa, the inhibition rate is plotted on the ordinate, and then the drug concentration at 50% inhibition is IC50. Key points: Dilute the drug twice, do more gradients, and do a dotted line chart.
Related links: MTT
Edited by Suzhou Yacoo Science Co., Ltd.