MTT Cell Proliferation and Cytotoxicity Detection Kit

ProductInformation

Description/Introduction

MTT is also called 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (Thiazolyl Blue Tetrazolium Bromide). Its molecular formula is C₁₈H₁₆BrN₅S, and the molecular weight is 414.32. 

MTT is commonly used for the detection of cytotoxicity and cell proliferation. The principle of the assay is that the enzyme succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT to water-insoluble blue-violet crystalline formazan, which is deposited in the cells, while the dead cells do not have such a chromogenic reaction. Dimethyl sulfoxide (DMSO) can dissolve formazan in the cells, and its light absorption value was measured by microplate absorbance reader, and the amount of MTT crystalline formazan formation was proportional to the number of cells within a certain cell number range.

In the traditional MTT assay, because of the generation of formazan is insoluble in water, it is necessary to remove the upper layer of reaction solution containing MTT substrate, and then add DMSO to dissolve the formazan, which is prone to cause poor repeatability and reproducibility of the experiment. This kit provides a special formazan dissolving agent, when the test does not need to remove the original MTT substrate-containing reaction solution, but directly add formazan dissolving agent, can dissolve formazan, so as to avoid the error caused by the removal of the original reaction solution.

Storage and Handling Conditions

Transport with wet ice; MTT solution, -20°C, protect from light; formazan dissolving agent, 4°C, protect from light; valid for 12 months.

Component

Assay Protocol / Procedures

1. For cell proliferation assay, it is recommended to seed 100 μL of 2000 cells per well; for cytotoxicity experiments, it is recommended to seed 100 μL of 5000 cells per well (depending on cell size and proliferation rate). Culture and transfection or drug pretreatment according to the needs of the experiment;

2. Add 10 μL of MTT solution to each well and incubate for 4 h in an incubator;

3. a. Add 100 μL of formazan dissolving agent to the well plate,

b. Tap the well plate to mix evenly

c. Incubate in the incubator for 2-4 hours to dissolve the formazan (Incubation time depends on the number of cells, can be observed under the microscope and can be detected when the formazan is dissolved).

4. Measure the absorbance of each well at 570 nm. If 570 nm filter is not available, the 560-600 nm filter can be used instead.

Note:

1. Use a 96-well plate for detection. If the cell culture time is long, it should be pay attention to the problem of evaporation. The circle around the 96-well plate is the easiest to evaporate. You can discard the circle and add PBS, water or culture medium instead. You can also ease the evaporation by placing the 96-well plate near the water source in the incubator.

2. In order to reduce the error caused by the experimental operation, it is recommended to set up duplicate wells to avoid the generation of air bubbles when adding samples or shaking and mixing.

3. The MTT solution is yellow. Before use, it should be placed at room temperature in the dark and water bath at 20-25°C for a while until it is completely melted before use. Prolonged exposure to light will cause failure, do not use if the solution is grey green.

4. If the formazan dissolving agent is precipitated, it can be dissolved in a water bath at room temperature or 37°C to promote dissolution, and then it can be used after dissolving and mixing.

5. For your health and safety, please wear a lab coat and disposable gloves when operating.

For Research Use Only!

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