MTT principle:
    The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystal formazan (Formazan) and deposit in cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at a wavelength of 490nm using a microplate reader. Within a certain number of cells, the amount of MTT crystals formed is proportional to the number of cells. According to the measured absorbance value (OD value) to determine the number of living cells, the larger the OD value, the stronger the cell activity (if the drug toxicity is measured, it means that the drug toxicity is less).
Adherent cells
1. Collect the cells in log phase, adjust the cell suspension concentration, add 100ul to each well, and plate to adjust the density of the cells to be tested to 1000-10000 wells (the marginal wells are filled with sterile PBS).
⒉, 5% CO2, incubate at 37 ° C, until the cell monolayer is covered with the bottom of the well (96-well flat bottom plate), add the drug with a concentration gradient. In principle, the drug can be added after the cells adhere to the wall, or two hours or half a day However, we often plate in the afternoon of the previous day and dosing the next morning. Generally 5-7 gradients, 100ul per well, set 3-5 multi-wells. It is recommended to set 5, otherwise it is difficult to reflect the real situation.
⒊, 5% CO2, incubate at 37 ° C for 16-48 hours, and observe under an inverted microscope.
4. Add 20ul MTT solution (5mg / ml, 0.5% MTT) to each well, and continue to culture for 4h. If the drug can react with MTT, discard the culture medium after centrifugation, carefully rinse with PBS 2-3 times, and then add the culture medium containing MTT.
5. Terminate the culture and carefully aspirate the culture medium in the wells.
6. Add 150ul of dimethyl sulfoxide to each well and shake on a shaker at low speed for 10min to fully dissolve the crystals. The absorbance of each well was measured at OD490nm of the enzyme-linked immunosorbent detector.
7. Set zeroing wells (medium, MTT, dimethyl sulfoxide) and control wells (cells, drug dissolving medium of the same concentration, culture solution, MTT, dimethyl sulfoxide) at the same time.
Determination of the maximum safe concentration of a drug:
 1. Take the highest concentration at which no lesions appear in the experimental wells, and the OD value of the medicated solution wells measured by the MTT method is not significantly lower than the OD value of the cell control wells as the maximum safe concentration.
2. Statistical processing
All data are expressed as mean ± standard deviation, and analysis of variance was performed using SPSS11.5. Finally, the average value of the experimental parallel wells was taken to calculate the percentage of cell destruction.
Destruction rate (%) = [(OD cell control well-OD administration well) / (OD cell control well-OD blank well)] × 100%.
The maximum drug concentration with a cell destruction rate of less than 5% was selected as the highest non-toxic dose (TC0) of the drug.