MTT Assay Kit

500T

Cell growth, cytotoxicity and proliferation assays

MTT reduction to formazan; colorimetric microplate assay

Absorbance: 490 nm.

Ship at low temperature; store at -20 °C protected from light. Solution B may be shipped and stored at room temperature.

1 year

MTT is widely used to assess cell growth. The underlying principle is that MTT can be reduced by dehydrogenases in the mitochondria of living cells to form dark-purple formazan crystals, whereas dead cells lack this activity. After the dark-purple formazan crystals are dissolved, their concentration can be determined by measuring the absorbance at 490 nm, and the viability of the cells can thereby be inferred: the more vigorous the cell proliferation, the higher the absorbance; the greater the cytotoxicity, the lower the absorbance.

1. A unique formazan dissolving solution is used, which dissolves formazan completely and reduces errors.

2. Low background, high sensitivity, wide linear range, and good reproducibility.

3. This product is sufficient for 500 wells (five 96-well cell culture plates) of microplate assays.

4. It can be used for bioactivity assays of biologically active factors, anti-tumor drug screening, cytotoxicity testing, determination of tumor radiosensitivity, etc.

The procedure below describes a cell cytotoxicity assay. Other applications are similar or even simpler (e.g., growth curve experiments); the procedure can be modified accordingly and will not be repeated here.

(I) Cell seeding

1. Digest a confluent monolayer of cells by conventional trypsin digestion and collect the cells into serum-containing culture medium.

2. Centrifuge at 200 × g for 5 minutes to collect the cell pellet.

3. Resuspend the cell pellet in culture medium to prepare a single-cell suspension and count the cells.

4. Dilute the cells to 2500–10000 cells/ml (to be decided according to the growth rate of the cells).

5. Transfer a sufficient amount of the cell suspension to culture dishes (to facilitate sampling with a multichannel pipette). The MTT assay for one 96-well plate requires about 20 ml of cell suspension.

6. Using a multichannel pipette, add 200 µL of cell suspension to the exact center of each well in columns 2–11 of the 96-well plate (for normal cells). For tumor cells, add 100 µL of tumor cell suspension and 100 µL of culture medium (total volume 200 µL). Note: the cells must be added to the exact center of the well; otherwise the cells will aggregate in the corners of the well and affect the assay.

7. Using the multichannel pipette, add culture medium in the same volume as the cell suspension to the wells in columns 1 and 12. The wells in column 1 will serve as the +medium −cells +MTT control (used for zeroing when measuring OD), and the medium added to column 12 serves to reduce the edge effect on the reactions in column 11.

8. Incubate at 37 °C and 5% CO₂ by conventional cell culture methods for 1–3 days so that the cells enter the exponential growth phase.

(II) Drug treatment

9. Dilute the drug in culture medium to 8 test concentrations (if the appropriate concentrations are unknown, a preliminary experiment is needed to determine them); generally, 3 parallel plates are required for one drug.

10. Remove the culture medium from the wells in columns 2 to 11 (10 columns in total; do not disturb the cells) while keeping the medium in the wells of columns 1 and 12.

11. Add 200 µL of fresh culture medium to the wells in columns 2 and 11 (2 columns in total); these wells will serve as the +medium +cells −drug control.

12. Add the 8 concentrations of the test drug to the wells in columns 3 to 10 (8 columns in total), adding one concentration per column.

13. Continue to incubate the 96-well plate at 37 °C and 5% CO₂ by conventional methods for a certain period of time; this period is the drug treatment time and is decided by the user.

14. At the end of the treatment, remove the culture medium from the wells in columns 2 to 11 (10 columns in total, all containing cells) and add 100 µL of fresh culture medium.

15. Change the medium daily so that the number of cells expands 2–3 fold (the time required varies with the cell type).

(III) Viable cell counting

16. At the end of the growth period, remove the culture medium from the wells in columns 1 to 11, then add 100 µL of fresh culture medium and 10 µL of Solution A (containing MTT). Wrap the 96-well plate in aluminum foil and continue culturing at 37 °C and 5% CO₂ for 4–8 hours. Note: Solution A solidifies at low temperatures; before use, allow it to stand at room temperature or warm it in a 20–25 °C water bath until completely dissolved, then mix well before use. MTT is carcinogenic — be sure to wear gloves.

17. Carefully aspirate and discard the medium in the wells (containing Solution A). Since the medium may affect light absorption, it is best to remove it as completely as possible.

18. Add 100 µL of Solution B to each well and shake on an orbital shaker at low speed for 10 minutes to fully dissolve the formazan crystals formed from MTT.

19. Because the product is unstable, immediately measure the absorbance at 490 nm on a microplate reader. Note: use the wells in column 1 (+medium −cells +MTT control) to zero the instrument.

20. Calculate the mean value of the replicates treated in the same way.

21. Plot a curve with the drug concentration on the x-axis and the absorbance on the y-axis. Because the absolute absorbance values of the different treatments differ greatly, they are generally converted into the growth inhibition rate (using the mean of the data from the drug-free wells in columns 2 and 11 as 100%) so that the IC50 can be calculated for comparing the effects of different drug treatments. Note: if a growth curve is being measured, plot time on the x-axis. A normal growth curve is generally S-shaped, and growth-promoting treatments give a steeper slope.

Ship at low temperature and store at -20 °C (Solution B may also be shipped and stored at room temperature). Valid for one year.

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications.