MTT assay, also known as MTT colorimetry, is a method to detect cell survival and growth.The detection principle is living cellsmitochondrionInSuccinate dehydrogenaseIt can reduce exogenous MTT to water insoluble blue purple crystalline formazan and deposit it in cells, while dead cells have no such function.Dimethyl sulfoxide(DMSO) energydissolutionMethane in cells, usingELISAAt 490nmwavelengthThe light absorption value can indirectly reflect the number of living cells.Within a certain range of cell numbers, the amount of MTT crystallization is proportional to the number of cells.This method has been widely used in somebiological activityFactor activity detection, large-scale anti-tumordrug screeningCytotoxicity test and tumor radiosensitivity test.It is characterized by high sensitivity and economy.
MTT is a method to detect cell survival and growth.MTT is a yellow compound, a dye that accepts hydrogen ions, and can act on the respiratory chain in the mitochondria of living cellsSuccinate dehydrogenaseandCytochrome CUnder the action of, exogenous MT was reduced to water insoluble blue purple crystalline formazan and deposited in cells, while dead cells had no such function.Dimethyl sulfoxide (DMSO) can dissolve methylzane in cells, and its light absorption value can be measured at 490nm wavelength with an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells.Within a certain range of cells, the amount of MT crystallization is proportional to the number of living cells[1]。
Preparation method
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MTT method
Generally, MTT in this methodconcentrationIs 5mg/ml.Therefore, 0.5 g MTT can be weighed and dissolved in 100 mlPhosphate buffer（PBS）Or phenol freeculture medium0.22 μ m filter membrane is used to removeBacteriaStore at 4 ℃ away from light.In the process of preparation and storage, it is better to wrap the container with aluminum foil paper.
Experimental steps
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Adherent cell
1. Collect logarithmic cells and adjust cell suspensionconcentration100ul is added to each hole, and planking is used to adjust the cells to be testeddensityTo 1000-10000 holes (for edge holessterilePBS filling).
2. Incubate with 5% CO2 at 37 ℃ until the cell monolayer is covered with the bottom of the hole (96 hole flat plate), addconcentrationGradientmedicineIn principle, we can add drugs after the cells adhere to the wall, or two hours, or half a day, but we often lay the board in the afternoon of the previous day, and add drugs in the morning of the next day. Generally, there are 5-7 gradients, 100ul per hole, and 3-5 multiple holes. It is recommended to set 5 holes, otherwise it is difficult to reflect the real situation
3. 5% CO2, incubation at 37 ℃ for 16-48 hours, invertedMicroscopeLower observation.
4. Add 20ul MTT solution (5mg/ml, i.e. 0.5% MTT) into each well and continue to culture for 4h.ifmedicineIt can react with MTT, and can be centrifuged before discardingCulture medium, carefully flush with PBS for 2-3 times, and then add MTT containingCulture medium。
5. Stop the cultivation and carefully suck out the holeCulture medium。
6. Add 150ul per holeDimethyl sulfoxide, settableUpper and lower speedoscillation10min, make the crystal fulldissolution。stayEnzyme-linked immunityThe absorbance value of each hole is measured at OD490nm of the detector.
1. CollectLogarithmic periodCells, Regulatory Cell Suspensionconcentration1×10six/Ml, in order to make up 1640 (none) of ①serum）culture medium40ul ；②Add Actinomycin D (toxicity) for 10ulCulture mediumDilute l g/ml, pre test is required to find the bestDilution，1:10-1:20)；③10 ul; ④Cell suspension 50ul (i.e. 5 × 10fourCell/hole), a total of 100ul is added to 96 hole plate (for edge holesterileWater filling).Each plate is set with control (add 100 μ (storage solution 100 1640).
2. Incubate at 37 ℃ with 5% CO2 for 16-48 hours, invertMicroscopeLower observation.
3. Add 10 ul MTT solution (5 mg/ml, i.e. 0.5% MTT) to each well and continue to culture for 4 h.（suspensionRecommended use of cellsWST-1, step 4) can be skipped after 4 h cultureELISAOD490nm measures thelight absorption value）
4、centrifugal(1000 revolutions x 10min), carefully suck out the supernatant, and add 100 ul per holeDimethyl sulfoxide, settableUpper and lower speedoscillation10 min to fully dissolve the crystal.The absorbance value of each hole was measured at OD490nm of ELISA.
5. At the same time, zero adjustment holes (culture medium, MTTDimethyl sulfoxide）, control well (cells, same concentrationmedicineDissolving medium, culture medium, MTT, dimethyl sulfoxide), each set with 3 double holes
shortcoming
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As the methylzan product produced by MTT reduction is insoluble in water, it can only be detected after being dissolved.This will not only increase the workload, but also affect the accuracy of the experimental results, and the organic solvents that dissolve methylzane will also harm the experimenter.
matters needing attention
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MTT method can only be used to detect the relative number and relative activity of cells, but cannot determine the absolute number of cells.When using the microplate reader to test the results, in order to ensure the linearity of the experimental results, the MTT absorbance should be within the range of 0-0.7.
MTT is generally best prepared on the spot. After filtration, it is effective within two weeks when stored in a dark place at 4 ℃, or 5mg/ml is prepared and stored at - 20 ℃ for long-term storageRepeated freezing and thawingIt is better to pack them in small doses and wrap them in light proof bags or black paper or tin foil to avoid decomposition.I usually put MTT powder into EP tubes and prepare it immediately when I use it. It is unnecessary to prepare so many MTT powder at once. Especially when MTT turns gray green, it can never be used again.
MTT is carcinogenic. Be careful when using it. If possible, it is better to use the transparent filmglove. The configured MTT needssterileMTT is very sensitive to bacteria;It doesn't matter if you don't avoid the light when adding time to the 96 hole plate. After all, the time is short, or you can turn off the lights on the console when you are worried
PBS for MTT preparationdissolution, also used by othersnormal saline60 ℃ water bath solution aid[2]。
