Cell proliferation assay: MTT method
The MTT assay, also known as the MTT colorimetric assay, can be used to: detect cell survival and growth.
Operation method
Cell proliferation assay: MTT assay
Principle
The MTT assay is based on the living cell metabolite reductant 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide, MTT thiazolium blue.MTT is a yellow color compound, a dye that accepts hydrogen ions, and acts on the respiratory chain in the mitochondria of the living cells. The tetrazolium ring cleaves under the action of succinate dehydrogenase and cytochrome C to form blue formazan crystals. The amount of formazan crystals produced is only proportional to the number of viable cells (the enzyme succinate dehydrogenase disappears in dead cells and MTT cannot be reduced). The reduced formazan crystals can be dissolved in an MTT dissolution solution containing 50% N,N-dimethylformamide and 20% sodium dodecamethylsulfonate (pH 4.7), and the optical density OD at 490 nm is measured using an enzyme marker to reflect the number of viable cells. It can also be solubilized in DMSO.
Materials and Instruments
Cell samples Move 1、Inoculation of cells Caveat 1、Choose the appropriate concentration of cell inoculation. 2， avoid serum interference: generally choose less than 10% of fetal bovine serum culture medium for the test. Try to suck out the residual culture solution in the well after color presentation. 3， set up a blank control: parallel to the test without adding cells and only add the culture medium of the blank control. Other test steps remain consistent, the final colorimetric blank to zero.MTT absorbance should be between 0-0. 7， beyond this range is not a linear relationship.IC50 is the half inhibition rate, meaning the concentration of the drug at 50% inhibition. Dilute the drug into different concentrations, then calculate the respective inhibition rate, use the concentration of the drug as the horizontal coordinate and the inhibition rate as the vertical coordinate to make a graph, and then get the concentration of the drug at 50% inhibition rate, which is the IC50. key point: dilute the drug by 2 times, make more gradients, and make a point line graph!ExampleThe concentration of each group is 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, the dilution is 10, the maximum concentration is 0.1, the inhibition rate is 0.95, 0.80, 0.65, 0.43, 0.21, 0.06. Substitute into the formula:Pm=0.95Pn=0.06P=0.95+0.80+0.65+0.43+0.21+0.06=3.1Xm=lg0.1=-1lgI=lg0.1/0.01=1lgIC50=-1-undefined (3.1-(3-0.95-0.06)/4)=-3.6025IC50=0.00025There is a formula for reference;lgIC50=Xm-I (P-(3-Pm-Pn)/4)Xm: lg maximum doseI: lg (maximum dose/adjacent dose)P: sum of positive response ratesPm: maximum positive reaction ratePn: minimum positive reaction rateInhibition rate = 1 - OD value of dosing group / OD value of control groupThe maximum and minimum positive reaction rate in the formula is the maximum and minimum inhibition rate.Example:How much 1640 medium, MTT and DMSO should be added to a 96-well plate to culture SMMC-7721 Liver Cancer for MTT measurement of cell viability? According to the book, 200ul of 1640, 20ul of MTT and 150ul of DMSO should be added to remove as much of the culture medium as possible before adding DMSO, so as to facilitate the dissolution of dirty particles by DMSO for the colorimetric assay.Generally 4000 cells per well is appropriate, both cell concentration in 20000 cells / ml, MTT add 20ul, the role of four hours after the supernatant is washed off, pay attention to do not wash off the dirty particles, and then add 150ul of DMSO per well, in the decolorization of shaking table shaking for 10 minutes, and then measure absorbance value.Generally it should be lower than IC50 to avoid too many cells killed by non-regulatory killing, causing too much debris detected by flow cytometry. I generally use 1/2-1/3 of the IC50, the action time is 36 h. The general tumor cell line blank treatment should be less than 1% of the modulation rate, after the drug is generally 5-10% (Annexin V), the sub-G0 peak of the cell cycle is more obvious. Common Problems Both MTT powder and solution need to be protected from light when stored and can be wrapped in aluminum foil. For more product details, please visit Aladdin Scientific website.
10% fetal calf serum MTT solution DMSO
96-well plate Centrifuge Enzyme immunoassay monitor
Prepare single cell suspension with culture medium containing 10% fetal calf serum and inoculate 1000-10000 cells per well into a 96-well plate, 200ul per well.
2、Culture of cells
Cultivate the cells under the same conditions as the general culture for 3-5 days (the incubation time can be decided according to the purpose of the test and the requirements).
3、Color presentation
After 3-5 days of incubation, add 20ul of MTT solution (5 mg/ml in PBS) to each well. Continue to incubate for 4 hours, terminate the incubation, and carefully aspirate the culture supernatant in the wells, or centrifuge and then aspirate the culture supernatant after centrifugation for suspended cells. Add 150ul of DMSO to each well and shake for 10 minutes to fully melt the crystals.
4. Colorimetry
Select the wavelength of 490nm, and measure the light absorption value of each well on the ELISA, record the results, and plot the cell growth curve with the time as the horizontal coordinate and the absorbance value as the vertical coordinate.