SPECIFICATION
MTT is widely used to detect cell growth, and its principle is that MTT can be reduced by dehydrogenase in the mitochondria of living cells to produce dark purple formazan crystals, while dead cells do not have this activity. After the deep purple formazan crystal is dissolved, its concentration can be determined by measuring the light absorption at 490nm wavelength, and thus inferring the vitality of cells. The more vigorous cell proliferation, the higher the absorbance; The greater the cytotoxicity, the lower the absorbance. The principle is as follows:MTT can be reduced by dehydrogenase in the mitochondria of living cells to generate deep purple formazan crystals, while dead cells do not have this activity principle
Product features:
1. Using a unique Formazan solution, it can fully dissolve Formazan and reduce errors.
2. Low background, high sensitivity, wide linear range, and good repeatability.
3. This product is sufficient for 500 times (5 96 well cell culture plates) of microplate testing.
4. It can be used for detecting the activity of bioactive factors, screening anti-tumor drugs, conducting cytotoxic tests, and determining tumor radiosensitivity.
Storage conditions: low-temperature transportation, -20 ℃ storage (but solution B can also be transported and stored at room temperature), with a validity period of one year.
Usage:
The following operation is a test for detecting cytotoxicity. Other applications are similar or simpler (such as growth curve test), and the operating steps can be slightly modified based on this, so it will not be repeated.
1. Inoculate Cells
1. Digest the confluence of monolayer cells using conventional pancreatic enzyme digestion method and collect them into serum-containing culture medium.
Centrifuge 2.200g for 5 minutes to collect cell precipitates.
3. Use culture medium to resuspend cell precipitation, prepare single cell suspension, and count.
4. Dilute the cells to 2.5 × 103 pieces/mL~5 × Between 10/mL (depending on the growth rate of the cell), if the degree of growth is not known, it can generally be diluted to 1 × 10 pieces/mL.
5. Transfer a sufficient amount of cell suspension to a culture dish (for easy sampling with a discharge gun). A 96 well plate MTT assay requires approximately 20mL of cell suspension.
6. Use a platoon gun to add 200 to the center of each hole in columns 2-11 of the 96-hole plate μ L cell suspension (for normal cells). If it is a tumor cell, add 100 μ L tumor cell suspension and 100 μ L medium (total volume 200 μ L) . Attention: Cells must be added to the center of the hole, otherwise they will gather at the corners of the hole and affect the experiment.
7. Add a culture medium of the same volume as the cell suspension to the first and 12th wells of the 96-well plate using a row gun. The wells in the first column will serve as the+medium cell+MTT control (used for zero adjustment during OD measurement), while the effect of adding culture medium in the 12th column is to reduce the influence of edge effects on the reactions in the 11th column.
8. Incubate the cells at 37 ℃ and 5% CO2 for 1-3 days using conventional cell culture methods to enter the exponential growth phase.
II. Drug Treatment
9. Dilute the drug to 8 test concentrations using a culture medium (if the test concentration is not known, a preliminary experiment is required to determine). Generally, three parallel plates need to be made for each drug.
10. Remove the culture medium from each well in columns 2 to 11 of 10 (do not touch cells), and retain the culture medium from each well in columns 1 and 12.
11. Add 200 to each hole in columns 2 and 11 of 2 μ L fresh culture medium, these wells will serve as control for+culture medium+cell drug.
12. Add 8 concentration gradients of the drug to be tested to each well in columns 3 to 10 of 8, with one concentration of drug added to each column.
13. Continue to incubate the 96 well plate under 37 ℃ and 5% CO2 conditions for a certain period of time according to the conventional method. This period is the time for drug treatment of cells, which is determined by the user.
14. After the treatment is completed, remove the culture medium from all wells in columns 2 to 11 (including 10 cells) and add 100 more μ L fresh culture medium.
15. Change culture every day to increase the number of cells by 2-3 times (the required time varies depending on the cells).
III. Survival Cell Count
16. At the end of growth, after removing the culture medium from the 1st to 11th wells, add 100 more μ L fresh medium and 10 μ L solution A (containing MTT component), wrap the 96 well plate in tin foil and continue to incubate at 37 ℃ and 5% CO2 for 4-8 hours. Attention: Solution A will solidify at low temperatures. Before use, please place at room temperature or take a water bath at 20-25 ℃ until fully dissolved. Shake well before use. MTT is carcinogenic and must be handled with gloves.
17. Carefully discard the culture medium (including solution A) in the well. As the culture medium may affect light absorption, it is best to remove it as much as possible.
18. Add 100 to each hole μ L solution B, shake at low speed on a shaking table for 10 minutes to fully dissolve the formazan crystals formed by MTT.
Due to the instability of the product, it is necessary to immediately select 490nm on the enzyme-linked immunosorbent assay to measure the absorbance.
Note: Use the first column of wells (+culture medium cells+MTT control) to zero.
20. Count the average value of each repetition processed similarly.
21. Draw a curve with drug concentration as the horizontal axis and absorbance as the vertical axis. Due to the significant difference in absolute absorbance values among different treatments, it is generally necessary to convert them into growth inhibition rates (using the average of the second and eleventh column data without drugs as 100%), in order to calculate IC50 and compare the effectiveness of various drug treatments. Note: If measuring the growth curve, use time as the horizontal axis. The normal growth curve generally presents an S-shaped shape, while those with promoting effects have an increased slope.