First, the problem should be clear before the experiment
1. Select the appropriate cell inoculation concentration.
In general, an inner adherent cell of a 96-well culture plate has about 105 cells when it is full. However, because the area of ​​different cells after attachment is very different, before the MTT test, the growth curve of the adherence rate, doubling time and different number of cells inoculated should be pre-tested to determine the inoculation of each well in the experiment. The number of cells and the culture time to ensure that the culture is terminated and the cells are overfilled. In this way, the linear relationship between the amount of MTT crystal formation and the number of cells can be ensured. Otherwise, the number of cells is too low and the sensitivity is reduced, too little difference is observed.
2. Setting of drug concentration.
Be sure to look at the literature more, and refer to the results of others to set a larger scope for the first screening. According to the results of the initial screening, the concentration and time range are narrowed and sieved. Remember! Otherwise, the time and concentration you may use is not the effective concentration and time of the drug at all.
3. The setting of the time point.
Measure the OD value at different time points, input the excel table, and finally get the change of the inhibition rate at different time points, draw the curve of the change, and when the curve becomes flat (to the plateau), the time point should be the best. Time point (because the most obvious inhibition of cell proliferation at this time).
4. Training time.
200 ul of culture medium is difficult to maintain for 68 h in the progenitor cells of 4 to 5 powers of 10, if the nutrition is not enough, the cells will gradually move from the proliferative phase to the G0 phase and tend to be stationary, affecting the results, we are Change the fluid at 48 h.
5. The MTT method can only measure the relative number and relative viability of cells, and the absolute number of cells cannot be determined.
When doing MTT, try to operate aseptically, because bacteria can also cause an increase in the MTT colorimetric OD value.
6. Theories are not necessarily correct.
It should be adjusted according to your actual situation.
7. Zero hole, control hole and dosing hole should be set during the experiment.
Zero wells were added to the medium, MTT, dimethyl sulfoxide. Cells, culture medium, MTT, and dimethyl sulfoxide were added to the control wells and the drug-added wells. The difference was that the control wells were mixed with the drug-dissolving medium, and the drug-added group was added with different concentrations of the drug.
8. Avoid serum interference.
When cells are cultured with 15% fetal bovine serum, high serum levels affect the light absorption of the test wells. Sensitivity will be tested as the test background increases. Therefore, a culture solution of less than 10% fetal calf serum is generally selected. After coloring, try to absorb the remaining culture solution in the culture well.
Second, the experimental steps
(a) adherent cells
1. Collect the log phase cells, adjust the cell suspension concentration, add 100 ul per well, and plate the cells to adjust the density to 1 000-10 000 wells (the edge wells are filled with sterile PBS).
2. Incubate with 5% CO 2 at 37 °C until the cell monolayer is covered with the bottom of the well (96-well flat bottom plate). Add a concentration gradient of the drug. In principle, the cells can be added after the cell is attached, or two hours, or half a day. Time, but we often lay the board the afternoon before, and add medicine the next morning. Generally 5-7 gradients, 100 ul per hole, set 3-5 duplicate holes. It is recommended to set 5, otherwise it is difficult to reflect the real situation.
3. Incubate for 5% CO 2 at 37 ° C for 16-48 hours and observe under an inverted microscope.
4. Add 20 ul of MTT solution (5 mg/ml, 0.5% MTT) to each well and continue to incubate for 4 h. If the drug can react with MTT, the culture solution can be discarded after centrifugation, carefully 2-3 times with PBS, and then the culture medium containing MTT is added.
5. Stop the culture and carefully remove the culture medium from the well.
6. Add 150 ul of dimethyl sulfoxide per well and shake on a shaker for 10 min at low speed to fully dissolve the crystals. The absorbance of each well was measured at the OD490 nm of the enzyme-linked immunosorbent assay.
7. Simultaneously set the zero adjustment hole (medium, MTT, dimethyl sulfoxide), control well (cell, same concentration of drug dissolution medium, culture solution, MTT, dimethyl sulfoxide).
(two) suspension cells
1. Collect log phase cells, adjust the cell suspension concentration to 1 × 106 / ml, and in sequence add 1 to 1640 (serum-free) medium 40 ul; 2 plus Actinomycin D (toxic) 10 ul diluted with culture medium ( Store solution 100 ug/ml, pre-test for optimal dilution, 1:10-1:20); 3 test substance 10 ul; 4 cell suspension 50 ul (ie 5×104 cells/well), total 100 ul Add to a 96-well plate (the edge wells are filled with sterile water). Set a control for each plate (plus 100 ul 1640).
2. Incubate at 37 ° C, 5% CO 2 for 16-48 hours and observe under an inverted microscope.
3. Add 10 ul of MTT solution (5 mg/ml, 0.5% MTT) to each well and continue to incubate for 4 h. (WST-1 is recommended for suspension cells, step 4 can be skipped after 4 hours of culture), and the absorbance of each well is measured by direct enzyme-linked immunosorbent assay OD570 nm (630 nm calibration).
4. Centrifuge (1000 rpm x 10 min), carefully aspirate the supernatant, add 100 ul of dimethyl sulfoxide per well, and shake on a shaker for 10 min at low speed to fully dissolve the crystals. The absorbance of each well was measured at OD570 nm (630 nm calibration).
5. Set the zero adjustment hole (medium, MTT, dimethyl sulfoxide), control well (cell, same concentration of drug dissolution medium, culture solution, MTT, dimethyl sulfoxide), set 3 sets for each group. hole.
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