1、 What is thiazole blue (MTT)
Thiazole blue, CAS No. 298-93-1, molecular formula c18h16brn5s, is a yellow or orange powder, melting point: 195 ℃ (decomposition). Slightly soluble in methanol, but hardly soluble in water or ethanol. It cannot be completely dissolved with ether, acetone or ethyl acetate. Soluble in DMSO. 5mg/ml stock solution is usually prepared.
2、 Detection principle of thiazole blue (MTT) colorimetry
Thiazole blue (MTT) colorimetry is a method to detect cell survival and growth. The principle of MTT experiment is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble blue purple crystal formazan and deposit it in cells. Dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve methyl Zan in cells. Its light absorption value is measured at 490nm wavelength by enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain range of cell number, the amount of MTT crystal formation is directly proportional to the number of cells. Judge the number of living cells according to the measured absorbance value (OD value). The greater the OD value, the stronger the cell activity (if the drug toxicity is measured, the less the drug toxicity is). This method has been widely used in the activity detection of some bioactive factors, large-scale anti-tumor drug screening, cytotoxicity test and tumor radiosensitivity determination. It is characterized by high sensitivity and economy.
3、 Application of thiazole blue (MTT)
MTT is usually used to determine cell proliferation, cell activity and cytotoxicity of drugs (including other treatments such as radiation) to cultured cells in vitro.
4、 Advantages and disadvantages of thiazole blue (MTT) colorimetry
Advantages: it is simple, fast, economical and does not use isotopes.
Disadvantages: as the methyl Zan product produced by the reduction of MTT is insoluble in water, it can be detected only after it is dissolved. The workload will increase, which will also affect the accuracy of the experimental results, and the organic solvent dissolving methyl Zan will also damage the health of the experimental personnel.
5、 Precautions for thiazole blue (MTT)
1. in the process of preparation and preservation, the container should preferably be wrapped with aluminum foil.
2. the prepared MTT needs to be sterile, and MTT is very sensitive to bacteria.
3. MTT is generally best to be used and prepared immediately. It is effective after being filtered and stored in the dark at 4 ℃ for two weeks, or it is prepared into 5mg/ml and stored at -20 ℃ for a long time to avoid repeated freezing and thawing. It is best to pack it in small doses and wrap it in a dark bag or black paper or tin foil paper to avoid decomposition. When MTT turns grey green, it can never be used again.
4. MTT is carcinogenic and should be used with care. If possible, it is best to wear transparent film gloves. Generally, the MTT concentration in this method is 5mg/ml. Therefore, 0.5g MTT can be weighed and dissolved in 100ml phosphate buffer (PBS) or phenol red free medium, and 0.22 μ M microporous membrane filtration to remove the bacteria in the solution, place it at 4 ℃ and store it away from light. In the process of preparation and preservation, it is better to wrap the container with aluminum foil.