MTT (tetrazolium salt) colorimetric method

Summary

The tetrazolium salt (MTT) colorimetric assay can be applied to (1) detection of cell survival and growth, (2) large-scale screening of antitumor drugs, (3) cytotoxicity assays as well as tumor radiosensitization assays, and (4) activity assays of bioactive factors.

Operation method

MTT colorimetric method

Principle

The enzyme dehydrogenase in living cells reduces the tetrazolium salt to a water-insoluble blue-violet product (formazan) that is deposited in the cells, whereas dead cells do not have this function. Dimethyl sulfoxide (DMSO) dissolves the blue-purple crystals deposited in the cells, and the color depth of the solution is proportional to the amount of formazan contained. The OD value was then determined by enzyme labeling.

Materials and Instruments

Cell samples
PBS DMSO trypsin Glycine buffer Dimethylmethylsulfoxide
Sampler 96-well culture plate Enzyme marker Culture plate

Move

I. Issues to be clarified before the experiment1. Choose the appropriate concentration of cell inoculation. In general, there are about ¹⁰⁵ cells when the wall-adherent cells in one of the 96-well culture plates are full grown. However, due to the great difference in the area of different cells after walling, therefore, before the MTT test, we should carry out the pre-test to detect the walling rate, doubling time and the growth curve under the condition of different inoculated cells, to determine the number of inoculated cells per well and the incubation time to ensure that the culture is terminated to the cell overfilling. In this way, we can ensure the linear relationship between the amount of MTT crystal formation and the number of cells. Otherwise, too many cells will reduce the sensitivity, and too few will not observe the difference.2. The setting of drug concentration. Must read more literature, reference to other people's results and then set a relatively large range of initial screening. According to the results of their own initial screening to narrow the concentration and time range and then fine screening. Remember! Otherwise, the time and concentration you use may not be the effective concentration and time of the drug at all.3. Time point setting. Measure the OD value at different time points, input the excel sheet, finally get the change of inhibition rate at different time points, draw the change curve, when the curve becomes flat (to the plateau period) that time point should be the best time point (because the inhibition of cell proliferation at this time shows the most obvious).4. Culturing time: 200 ul of culture medium is difficult to maintain 68 h for the proliferating cells of 4~5 times 10, and if the nutrition is not enough, the cells will gradually tend to the G0 phase from the proliferating phase to the quiescent phase, which will affect the results, so we changed the culture medium at 48 h. We also changed the culture medium at 48 h.5. MTT method can only determine the relative number of cells and relative viability, but not the absolute number of cells. When doing MTT, try to operate as aseptically as possible, because bacteria can also cause the MTT colorimetric OD value to increase.6. Theory may not always be right. It should be adjusted according to your actual situation.7. The experiment should be set up with a zeroing hole, a control hole, and a dosing hole. Add culture medium, MTT and dimethyl sulfoxide to the zeroing hole. Control and dosing holes should be added with cells, culture medium, MTT, dimethyl sulfoxide, the difference is that the control holes are added with the medium for dissolving drugs, while the dosing group is added with different concentrations of drugs.8. Avoid serum interference. When cells are cultured with culture medium containing 15% fetal bovine serum, high serum material will affect the light absorption value of the test wells. Due to the increase of the test background, it will test sensitivity. Therefore, a culture solution less than 10% fetal bovine serum is usually selected for this purpose. After color presentation, try to aspirate the residual culture solution in the culture wells.
II. Adherent cells1. Collect log phase cells, adjust the concentration of cell suspension, add 100 ul per well, spread the plate so that the cells to be tested are densified to 1,000-10,000 wells, (the edge of the wells filled with sterile PBS).2. 5% _CO2, 37 ℃ incubation, until the cell monolayer spread to the bottom of the wells (96-well flat-bottomed plate), add the concentration gradient of the drug, in principle, the drug can be added after the cell apposition, or two hours, or half a day time, but we often in the afternoon of the previous day to lay the plate, and add the drug in the morning of the next day. Generally 5-7 gradients, 100 ul per well, set up 3-5 replicate wells. It is recommended to set 5, otherwise it is difficult to reflect the real situation.3. 5% _CO2, incubate at 37℃ for 16-48 hours, observe under inverted microscope.4. Add 20 ul MTT solution (5 mg/ml, i.e. 0.5% MTT) to each well, and continue to incubate for 4 h. If the drug and MTT can react, centrifuge and discard the culture solution first, and then add the MTT-containing culture solution after rinsing it carefully with PBS for 2-3 times.5. Terminate the incubation and carefully aspirate the culture medium from the wells.6. Add 150 ul of dimethyl sulfoxide to each well, and put it on a shaker with low-speed shaking for 10 min, so as to fully dissolve the crystals. Measure the absorbance value of each well at OD490nm of enzyme immunoassay detector.7. At the same time, set up zeroing wells (culture medium, MTT, dimethyl sulfoxide), control wells (cells, the same concentration of drug dissolution medium, culture medium, MTT, dimethyl sulfoxide).III. Suspension cells1. Collect the log phase cells, adjust the concentration of cell suspension ^1x106/ml, and sequentially place the

(1) Make up 40 ul of 1640 (serum free) medium;

(2) Add Actinomycin D (toxic) 10 ul diluted with culture medium (storage solution 100 ug/ml, need to be pre-tested to find the optimal dilution, 1:10-1:20);
(3) 10 ul of assay is required;
(4) 50 ul of cell suspension (i.e. ^{5x104 cells/well} ), total 100 ul added to 96-well plate (edge wells filled with sterile water). A control was set up for each plate (100 ul of 1640 was added).2. Incubate at 37℃ with 5% _CO2 for 16-48 hours and observe under inverted microscope.3. Add 10 ul MTT solution (5 mg/ml, i.e. 0.5% MTT) to each well, and continue to incubate for 4 h. (WST-1 is recommended for suspension cells, and step 4 can be skipped after 4 h of incubation.) Measure the absorbance value of each well by direct ELISA at OD570 nm (630 nm calibration).4. Centrifuge (1000 rpm x 10 min), carefully aspirate off the supernatant, add 100 ul dimethyl sulfoxide to each well, and set on a shaker with low speed shaking for 10 min to fully dissolve the crystalline material. Measure the absorbance value of each well at OD570nm (630nm calibration) of the ELISA.5. Simultaneously set up zeroing wells (culture medium, MTT, dimethyl sulfoxide), control wells (cells, the same concentration of drug dissolution medium, culture medium, MTT, dimethyl sulfoxide), and set up 3 duplicate wells in each group.IV. Preparation of MTTMTT is generally best used now, filtered 4oC light preservation is effective for two weeks, or formulated as 5mg/ml stored at -20 degrees for long-term preservation, to avoid repeated freezing and thawing, it is best to small doses divided into packages, with light bags or black paper, tin foil wrapped to avoid decomposition of light. I usually dispense the MTT powder in EP tubes and add it directly to the culture plate when I use it, there is no need to dispense so much at once, especially when the MTT turns grayish-green, then it should never be used again.MTT is carcinogenic, so be careful when you use it, and if possible, you'd better wear those transparent gloves. The MTT needs to be sterile, MTT is very sensitive to bacteria; it is okay to add MTT to 96-well plates without protecting it from light, after all, the time is relatively short, or you can turn off the light on the operating table if you are not sure.When preparing MTT, use PBS to dissolve, some people also use saline to prepare, 60 ℃ water bath to help dissolve.1. PBS formula(1) NaCl: 8g.(2) KCl 0.2: g.(3) _Na2HPO4: 1.44 g.(4) _KH2PO4: 0.24 g.(5) Adjust pH 7.4.(6) Volume 1 L.V. Inoculation of cells (plate spreading)Cells should not be used after 30 generations, because the state is not good; the culture plate should be good (preferably imported plate), bad plate or reuse of the plate can only be used for pre-experiment.When inoculating, it is better to inoculate according to the density figured out in the pre-test, because when the cell density is around 10000/ml, the OD value of the measured interval, i.e. the cell inhibition rate (or the value-added rate) shows the best linear relationship, and the result is the most trustworthy. If the cells are spread too thinly, the killing will not be obvious, and if they are too dense, the cells may die because the cells grow too fast and do not have enough nutrients, which will eventually lead to death. And if the cells are too dense or too few, the proliferation will be too fast or too slow, and the linear relationship between their value-added rate is not good. Therefore, MTT cell density is mostly 10000/ml, 100 ul/well.The cell density should be determined according to the characteristics of different cells. If the drug you make has a stimulating effect on the cells then take a smaller cell concentration, if the drug you make has an inhibitory effect on the cells then take a larger cell concentration, so that the difference with the control is more obvious and the data is better. The number of cells per well can be ¹⁰⁵ for suspension cells and ^103-104 for adherent cells _^.

Caveat

1. Choose the appropriate concentration of cell inoculation.

2. Avoid serum interference: generally choose less than 10% fetal bovine serum culture medium for the test. Try to suck out the residual culture solution in the wells 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 the same, the final colorimetric blank to zero.MTT experiment absorbance should be between 0-0.

7， beyond this range is not a linear relationship, IC50 is the half inhibition rate, meaning that the inhibition rate of 50% when the concentration of the drug. Dilute the drug into different concentrations, then calculate the respective inhibition rate, the concentration of the drug as the horizontal coordinate, the inhibition rate as the vertical coordinate of the graph, and then get the concentration of the drug at 50% inhibition rate, that is, the IC50. key points: the drug is diluted by 2 times, and more gradients, and do some point line graphs can be!An example:Each group concentration 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, dilution times of 10, the maximum concentration of 0.1, the inhibition rate of 0.95, 0.80, 0.65, 0.43, 0.21, 0.06. Substitution of 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=0.00025Refer to the formula:lgIC50=Xm-I(P-(3-Pm-Pn)/4)Xm:lg maximum doseI:lg(maximal dose/critical dose)P:sum of positive reaction ratePm: maximum positive reaction ratePn: minimum positive response rateInhibition rate = 1 - OD value of dosing group / OD value of control groupThe maximum and minimum positive response rate in the formula is the maximum and minimum inhibition rate.Example:Cultivate SMMC-7721 hepatocellular carcinoma in 96-well plate to do MTT to measure cell viability, how much 1640 medium should be added, how much MTT and DMSO are suitable according to the book to add 200 ul 1640, 20 u l MTT, 150 ul DMSO to add DMSO to try to remove the culture medium before adding DMSO, so as to facilitate the dissolution of dirty particles in the colorimetric assay with DMSO. 4000 cells are usually suitable for each well, and the cell concentration is also suitable. 4000 cells per well is appropriate, both cell concentration in 20000 cells / ml, MTT add 20 ul, the role of four hours after the supernatant is washed off, pay attention to do not wash off the dirty particles, and then add 150 ul DMSO per well, in the decolorization shaker shaking for 10 minutes, and then measure the absorbance value.

Common Problems

I. Summary of experience

1. First of all, the inoculation density of cells must not be too large, generally about 1000 cells per well is enough, I think it is better to have less than more. 10,000/well is too high, so even if the drug has an effect, the MTT method will not show it. Especially for tumor cells, 10000/well is too high, so that even if the drug has an effect, the MTT method is not shown, the best point plate concentration in 4000-5000/well, too little if the SD value will be very large.

2. MTT itself is a relatively coarse experiment, the proliferation rate of about 10% fluctuations are not strange. Especially for novices, 20% fluctuation is also common, so it is likely to be caused by technical reasons, especially the seed plate technology must be over the top.

3, I do is the tumor cell MTT experiment, this kind of cell growth is very fast at first I used 100000/ML concentration to inoculate, the result of the cell growth is too full result is no gradient and no linear relationship. Later, I adjusted the concentration and used 40000~80,000/ML concentration to do MTT experiment, and found that the better result was 60000~70,000/ML concentration group. With 40,000/M concentration group, due to the small number of cells, the gradient of drug action is still there, just not a good linear relationship. Also, depending on the growth rate of the cells and the characteristics of the drug (time-dependent and concentration-dependent drugs), it is necessary to determine whether the incubation time should be 48 hours or 72 hours.

4. Pay attention to the cell suspension must be mixed, to avoid the cell precipitation, resulting in the number of cells in each well is not the same, can be every few to be mixed again. Sampler operation should be skillful, try to avoid human error. Although the pipette is much more accurate than the pipette, if the operation is not skillful, the CV will be around 8%. In addition, blowing apart too many times will affect cell viability. So be skillful with the shoes and get on the plate quickly.

Second, the experimental experience sharing

1. The total amount of suspension should not be too much when blowing, 3-4 times of the pipette volume, it may be easier to mix. 10 ml centrifuge tube should be filled with 3-4 ml of suspension: too little suspension is easy to blow up a lot of bubbles, and too much suspension is not easy to blow up into a single-cell suspension.

2. The suction volume of the straw should be around 1 ml: if the suction volume is too high, a lot of liquid will be sucked up at once, and there will be very little liquid left in the pipe, which makes the blowing easy to blister; if the suction volume is too low, the blowing will not be strong enough, and the blowing will be uneven. If you use a pipette with a suction volume of 1 ml or more, a total volume of about 5 ml would be beneficial.

3. Suction should be at the bottom of the suspension, and then lift up a little, but don't leave the surface of the liquid when blowing down, otherwise it is easy to blow bubbles.

4. Blow about 100 times, it can be blown evenly (some people think that adding cells before blowing 30-50 times is basically almost uniform. When adding cells, blow 3 times per inoculation of 2 holes repeatedly, and after each blow 3 times the tip of the gun hangs down with the cell suspension for 5 seconds, and then aspirate the suspension at a certain speed.)

5. Do not be too fast when adding cells to each well with the lance tip, otherwise you will find that the cells will gather in a pile at the bottom of the well due to the impulse of the lance tip at the moment of addition, usually in the center of the bottom of the well and very little at the periphery, and this uneven dispersion will produce contact inhibition and affect the growth of the cells. So the speed should not be too fast or too slow. I used to hold each plate flat in my hand after adding, move 3 times to the left, 3 times to the right, and 3 times back and forth in return, the purpose is to make the cells can be dispersed more evenly. (Plate spreading technique is the key to MTT experiment, but also the foundation, must be practiced. Once a student used a vortex oscillator to mix the cells, and finally all the cells died, it is recommended not to use this method to mix the cells.

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