First, what is MTT?
MTT, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, is a yellow-colored dye commonly used in biological assays. It is also known as thiazole blue and is widely used in cell viability and cytotoxicity studies.
Second, what is the MTT method used for?
The MTT assay is a popular technique to assess cell survival, proliferation, and drug toxicity. It helps determine how well cells are functioning after exposure to different treatments, such as drugs or radiation.
Third, why can MTT be used for these purposes?
The principle behind the MTT assay lies in the ability of living cells to reduce MTT into an insoluble blue-purple compound called formazan. This reaction occurs due to the activity of mitochondrial enzymes like succinate dehydrogenase. Dead cells cannot perform this reduction. After incubation, the formazan crystals are dissolved using solvents like DMSO or a triple solution, and the absorbance at 490 nm (or sometimes 570 nm) is measured. The higher the absorbance, the more viable cells there are, making it a reliable indicator of cell activity.
Fourth, what materials are needed for the experiment?
1. MTT Solution Preparation: Typically, MTT is prepared at a concentration of 5 mg/mL using PBS or physiological saline. Small packages (like 100 mg) should be dissolved completely, filtered, and stored in dark containers at -20°C. Large quantities can be weighed and stored similarly. Always handle with care, as MTT is potentially carcinogenic.
2. DMSO or Triple Solution: DMSO is commonly used to dissolve formazan crystals, but it is toxic and requires careful handling. Alternatively, a triple solution containing SDS, isobutanol, and HCl can be used, though it dissolves more slowly and must be warmed to room temperature before use.
Fifth, the experimental steps
1. Prepare a cell suspension from log-phase cells, adjusting the concentration to around 5–10×10ⴠcells/mL. Use a hemocytometer to count and adjust the cell density accordingly.
2. Seed the cells into a 96-well plate, adding 100 µL per well. Include control wells and duplicate samples for accuracy.
3. Incubate the cells for 24–48 hours, depending on the experiment, then add the test drug in various concentrations.
4. After 4 hours of incubation with MTT, remove the culture medium and add the dissolution solution (DMSO or triple solution).
5. Incubate further until all formazan crystals are dissolved, then measure the absorbance at 490 nm using a microplate reader.
Sixth, how to analyze the results
To calculate IC50, one common method is the improved寇 method: lgIC50 = Xm - I × [P - (3 - Pm - Pn)/4]. Here, Xm is the logarithm of the maximum dose, I is the logarithmic difference between doses, and P, Pm, and Pn represent the cumulative positive response rates. This approach allows for accurate determination of the half-maximal inhibitory concentration of a drug.
It’s important to ensure that all steps are carefully followed, especially when preparing solutions and handling MTT, which can be hazardous. Proper controls and replicates are essential for reliable results. Always work in a fume hood and wear appropriate protective gear, including gloves and goggles.
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