The MTT assay is a common method for cell growth detection by MTT (chemical name 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide, trade name thiazolyl blue). This method is widely used because of its high sensitivity, good reproducibility, easy operation, economy and safety.
Principle
The basic principle of the MTT assay for cell growth is to utilize MTT's ability to accept hydrogen atoms for color development. Mitochondria in living cells have succinate dehydrogenase, which reduces exogenous MTT to insoluble blue-violet crystals, whereas dead cells lack mitochondrial activity and therefore do not have this activity.
The blue-purple crystals can be dissolved in dimethyl sulfoxide, and the light absorption value can be detected at 490 nm by using an enzyme marker, and the number of cells can be judged according to the absorption value. Within a certain number of cells, the absorption value is proportional to the number of cells. Recent studies have proved that the optimal absorption of blue-violet crystalline material is 570 nm.
Operation method
Cytotoxicity and proliferation assay by MTT method
Principle
Some dehydrogenases in the mitochondria of living cells are able to reduce exogenous MTT to insoluble blue-violet crystals, while dead cells do not have this activity. The blue-purple crystals can be dissolved in dimethyl sulfoxide (DMSO), and cell toxicity and proliferation can be quantified by measuring the absorbance (OD) at 490 nm with an enzyme marker. Cell proliferation is directly proportional to absorbance, and cytotoxicity is inversely proportional to absorbance. The absorbance value at 570 nm is measured when using the established kits.
Materials and Instruments
Cells, 96-well plates, pipette gun, tip, 15 ml centrifuge tube, PBS, trypsin, cell culture medium, enzyme marker, dimethyl sulfoxide, MTT Move 1, according to the instructions for the preparation of MTT solution, using PBS to prepare the final concentration of 5 mg/ml, after preparation can be appropriately divided and put -20 ℃ light preservation. 2. Inoculate the appropriate concentration of cells into a 96-well plate and process according to normal experimental requirements. 3、After incubation, add 10 μl of MTT solution to each well and incubate at 37 ℃ for 4 hours. 4. Carefully remove the supernatant from the wells (centrifugation is required for suspended cells, and the supernatant should be discarded after centrifugation), add 150 μl of DMSO, and then shake the wells at a low speed for 10 min, so as to dissolve the purple crystals. The OD value of each well was measured at 490 nm on an ELISA instrument and the results were recorded. 5. In addition to DMSO, a well-established MTT kit can also be chosen for the test. 100 μl of crystallization solution is added to each well, and incubation is continued for 3~4 hours in a cell culture incubator until all the crystals are melted. Measure the absorbance at 570 nm. Caveat 1. The OD value of the ELISA test is only linearly related to the number of cells at the appropriate cell concentration. Therefore, in the experiment, you must choose the appropriate concentration of cell inoculation, generally about 1,000~5,000 cells per well of 96-well plate, and pay attention to the inoculation of the plate should be mixed well, to prevent the cells from clumping and uneven distribution. 2. Serum substances will interfere with the OD value, therefore, after color development, absorb the residual medium in the wells as much as possible. 3. Set a blank control. Set up a blank control well without cells but only with medium in parallel with the test wells, and keep the other experimental steps the same. During colorimetry, the blank wells will be used for zero adjustment. 4. If the incubation time of 96-well plate is long, pay attention to the evaporation problem. Discard the surrounding circle and replace it with PBS or culture medium. 5. MTT should be prepared on the spot and avoid repeated freezing and thawing. It is easy to solidify at low temperature and should be thawed before use. 6. Before determining the OD value, make sure that there are no air bubbles in the cell well plate. 7. After adding MTT to generate crystals, be careful not to remove the crystals when removing the supernatant. 8. The crystals must be completely melted before measuring the OD value. For more product details, please visit Aladdin Scientific website.