MTT is a powdered chemical called 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide. The Chinese chemical name is 3-(4,5-dimethylthiazole. -2)-2,5-Diphenyltetrazolium bromide, trade name: Thiazole Blue, is a yellow color dye. MTT has two main purposes: (1) the determination of cytotoxicity of drugs in vitro (including other treatments such as radiation); (2) cell proliferation and cell viability assay.
The MTT method can measure the proliferative response of cells simply, quickly and accurately, and its price is low, which has become one of the first choice methods for detecting cell activity in various laboratories. The detection principle is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan (Formazan) and deposit in cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its light absorption value is measured by a microplate reader at a wavelength of 490 nm. The amount of MTT crystal formation is proportional to the number of cells in a certain number of cells. The number of viable cells is judged based on the measured absorbance value (OD value). The larger the OD value, the stronger the cell activity (if the drug toxicity is measured, the drug toxicity is smaller).
First, the preparation of MTT solution
1. Preparation of MTT solution The final concentration of MTT is usually 5 mg/ml, and PBS or physiological saline should be used as a solvent.
The general MTT package on the market is 100 mg, 250 mg or 1 g. For the small package of 100mg, the manufacturer puts the MTT into the small tube. Personally, it is recommended not to use the balance to weigh the package. Instead, it should be formulated into a solution at one time, such as 100mg dissolved in 20ml PBS. Specifically: pre-mix 50ml PBS in a 50ml centrifuge tube (if it can be replaced with a culture flask), take 500-1000ul PBS into the small tube containing MTT, blow it several times, then transfer it to a 50ml centrifuge tube, then mix again. uniform. It can be repeated several times so that the MTT in the small tube does not remain in the tube. After thoroughly mixing the MTT, the bacteria in the solution were removed by filtration through a 0.22 μm filter, and stored in a dark place (protected from a light-proof bag or black paper or tin foil) and stored at -20 ° C for a long time. According to the cell culture plate, 10 ul is required for each hole. Generally, about 1 ml is required for each 96-well plate. Therefore, 1 ml of each tube can be considered for dispensing. For large packaging, a part of the solution can be weighed according to the above method. Some people also dispense the powder into the EP tube, and when it is used, it is added directly to the culture plate.
2, note:
(1) During the preparation and storage process, the container is preferably wrapped in aluminum foil.
(2) The formulated MTT needs to be sterile, and MTT is very sensitive to bacteria.
(3) MTT is generally best used now, filtered at 4 ° C in the dark for 2 weeks, or 5mg / ml stored at -20 degrees for long-term preservation, to avoid repeated freezing and thawing, preferably small dose dispensing, Wrap it in dark light or black paper or tin foil to avoid decomposition. When the MTT turns grayish green, it can never be used again.
(4) MTT is carcinogenic and should be used with care.
Second, MTT formazan solution
1, dimethyl sulfoxide DMSO: can be directly dissolved, without preparation, easy to use, fast dissolution, but more toxic to humans, and need to remove the original culture solution, in the process of removing the culture solution, may remove the crystallization , resulting in unstable results.
2, triple solution: SDS10g, isobutanol 5ml, 10M HCl 0.1ml, dissolved in double distilled water to form a 100ml solution, the solution does not need to remove the original medium, but dissolve slowly. Since the solution contains SDS and is easily crystallized at a low temperature, it must be taken to room temperature several hours before use, and the SDS crystals are all dissolved and used.
Third, the MTT method experimental steps
1. Trypsin digested the log phase cells, centrifuged and collected after centrifugation to prepare a cell suspension, and adjust the concentration of the cells to 5-10×10 4 /ml.
2. After preparing the cell suspension, mix gently, add 100 ul per well, so that the density of the cells to be tested is 5000-10000/well (the edge wells are filled with sterile PBS).
Note: Since the cells still need to settle after mixing, the mixture should be mixed repeatedly several times during the inoculation process, such as mixing every 6 wells to ensure that the density of the inoculated cells is exactly the same between the wells. This is crucial for the results of MTT.
3. Place the inoculated cell culture plate in an incubator and incubate the cell monolayer to the bottom of the well (96-well flat bottom plate) and add a concentration gradient of the drug. In principle, the cells can be added after the wall is attached (two hours - half a day), but we often plate the plate the previous day and add the drug the next morning. Generally 5-7 gradients, 100ul per hole, 3-5 duplicate holes, it is recommended to set 6, otherwise it is difficult to reflect the real situation.
Note: For dosing, the drug is added directly to the 96-well plate in different volumes to form a concentration gradient. However, I believe that the different concentrations of the drug should be prepared in the EP tube, and then the culture supernatant in the 96-well plate should be removed (can be removed by a lance) and then 100 ul of medium containing different concentrations of the drug can be added to ensure the drug. The concentration is accurate. In addition, it should be noted that if this method is used, do not take all the culture medium of the 96-well plate and then add the drug. It should be added immediately after the part is sucked to avoid cell death caused by the drying of the 96-well plate.
4, 5% CO2, incubate at 37 ° C for 16-48h, observe the effect of the drug under an inverted microscope.
5. Add 10 ul of MTT solution (5 mg/ml, ie 0.5% MTT) to each well and continue to culture for 4 h.
6. Terminate the culture and prepare to dissolve the crystals.
There are two ways to dissolve crystals, dissolved in DMSO:
1) Dissolution in DMSO: After MTT was added for 4 hours, the crystals were sufficiently formed. The supernatant is removed and the process must be careful not to remove the Formazan crystals. Some people directly use the pipette to remove the supernatant. It is also recommended to lay a few sheets of filter paper on the tabletop, then gently invert the 96-well plate so that the supernatant is sucked away by the filter paper to reduce the loss of results.
2) Add 150 ul of dimethyl sulfoxide to each well and shake at low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance of each well was measured at an enzyme-linked immunosorbent detector at OD490nm.
Another method, using a triple solution:
1) After MTT was added for 4 hours, the crystals were sufficiently formed. In this way, the cell supernatant can be removed without adding 100 ul of solution directly to each well. (This solution contains SDS and is easy to crystallize when stored at a low temperature. Therefore, it must be taken to room temperature several hours before use, and the SDS crystals are completely dissolved before use.)
2) Incubate in an incubator for 4-6 hours, observe under the microscope, and measure the absorbance at 570 nm after all the crystals are dissolved. Usually, it is incubated at 37 ° C for about 4 hours, and the purple crystals are all dissolved. If the purple crystals are smaller and less, the dissolution time will be shorter. If the purple crystals are larger, the dissolution time will be longer.
7. Simultaneously set the zero adjustment hole (medium, MTT, dimethyl sulfoxide), control well (cell, the same concentration of drug dissolution medium, culture solution, MTT, dimethyl sulfoxide).
Fourth, MTT results analysis
How to calculate IC50 - improved 寇 method: lgIC50=Xm-I[P-(3-Pm-Pn)/4]
Xm: lg maximum dose
I: lg (maximum dose / adjacent dose)
P0: sum of positive reaction rates
Pm: maximum positive response rate
Pn: minimum positive response rate
Formazan formed by the MTT method is water-insoluble and needs to be dissolved by an organic solvent. Since the small portion of the Formazan may be carried away during the de-clearing operation, the repeatability is sometimes slightly poor. To solve this problem, the researchers have developed a variety of water-soluble tetrazolium salts: such as XTT, CCK-8 (WST-8).
Cell viability detection related kits:
MTT cell proliferation and cytotoxicity test kit
CCK-8 cell proliferation and cytotoxicity test kit
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