Thiazolyl Blue (MTT) is a colorimetric compound extensively utilized to measure cell proliferation, transitioning from a yellow color to purple formazan in living cells.

Instructions
1. Reagent preparation: First, dissolve MTT in an appropriate solvent (usually PBS or saline), usually at a concentration of 0.5 mg/mL to 5 mg/mL, and the specific concentration can be adjusted according to the experimental needs. Remove insoluble matter by filtration or centrifugation to ensure that the solution is pure.
2. Operation steps
1. Cell treatment: Inoculate cells in a 96-well plate or other appropriate culture plate and culture to a suitable density (e.g. 1 × 10⁴ to 1 × 10⁶ cells/well).
After the cell culture is completed, add a certain volume of MTT solution (usually 10 µL to 50 µL) to ensure that the final concentration in each well is 0.5 mg/mL to 1 mg/mL.
2. Incubate the cells with MTT, usually for 2 to 4 hours, and the specific time can be adjusted according to the experimental requirements.
3. Reaction treatment: After the incubation, remove the culture medium and add an appropriate amount of solvent (such as DMSO or isopropanol) to dissolve the purple formazan crystals formed. DMSO is a common solvent, and 100 µL to 200 µL of DMSO is usually added. Gently shake the plate to ensure complete dissolution.
4. Colorimetric assay: Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 570 nm (or 590 nm). The purple formazan product is proportional to the number of viable cells, so the higher the absorbance value, the stronger the cell activity. The absorbance of each sample is obtained by calculating the difference in absorbance with the blank well.
5. Data analysis:
1) Based on the absorbance data, the proliferation rate or survival rate of the cells can be calculated.
2) The effect of drugs or chemical reagents on cells is often calculated by comparing the OD values ​​between the control group and the treatment group.
3) The standard curve method can be used to further quantify the cell survival rate.
Notes
1. Cell density: The inoculation density of cells should be appropriate. If the cells are too dense, it may lead to excessive reaction and affect the experimental results; if the cell density is too low, it may not produce enough signal.
2. Solubility: When using DMSO or other solvents, make sure that the solvent is not toxic to the cells.
3. Time control: Too long or too short incubation time may affect the MTT reduction reaction, so the time needs to be optimized according to the cell type and experimental purpose.