MTT-based cytotoxicity assay
MTT-based cytotoxicity assay
It is mainly used to detect the killing activity of effector cells on target cells.
Operation method
MTT-based cytotoxicity assay
Principle
Cells in the exponential growth phase are exposed to cytotoxic drugs; the duration of exposure is usually determined by the time required to produce maximal damage, but it is also influenced by the stability of the drug. After the drug is withdrawn, the cells are allowed to proliferate for an additional 2 to 3 population doubling times (PDT ), which distinguishes the surviving cells that are able to proliferate from those that are not. The number of viable cells is then determined by MTT dye reduction reaction. As long as the MTT-stained product is dissolved in a suitable solvent, it can be quantified spectrophotometrically.
Materials and Instruments
Cell Samples Move Material: Non-sterile Caveat 1. Strict sterilization. Use three sets of instruments to take materials. 2. Strictly carry out aseptic operation to prevent bacterial, fungal and mycoplasma contamination and avoid chemical substance contamination. 3. Flame sterilize the mouth of the bottle and the pipette before aspirating the liquid; always use Hank'S liquid to cool the pipette after being flame sterilized. Prevent scalding and scalded cells. When aspirating liquids, avoid bottle mouth and pipette contact collision. For more product details, please visit Aladdin Scientific website.
Trypsin MTT Sorensen Glycine Buffer
Microtitration plates Micropipettes Petri dishes
Sterile
Growth solution.
Trypsin (0.25% + EDTA , 1 mmol/L, dissolved in PBSA)
MTT : 3-(4,5-dimethyl-2-thiazole)-2, 5-dimethylphenyltetrazolium bromide, 50 mg/m l, filtered, sterilized.
Sorensen glycine buffer (0.1 mol/L glycine, 0.1 mol/L, NaC l, pH adjusted to 10.5 with 1 mol/L Na(OH))
Microtitration plate (Iwaki)
Micropipettes, preferably in autoclaved tip cases
5 cm and 9 cm Petri dishes (non-TC treated) or reservoirs (Corning)
30 ml and 100 ml universal containers or tubes 
Plastic boxes (non-toxic polystyrene for plates)
Multi-channel sampler
Dimethyl sulfoxide (DMSO) DMS( ) Dispenser: Labsystems Microplate Dispenser (Cat No 5840127, Thermo Electron)
ELISA plate reader (Molecular Devices, with SOFT max PRO; see Appendix II)
Plate rack for centrifugation (for suspension of growing cells)
Procedure
Inoculate cells
1. Digest confluent monolayers of cells with trypsin and collect cells into serum-containing medium.
2. Centrifuge the cell suspension (5 min , 200 g ) to deposit the cells. Resuspend the cells in medium and count.
3. Dilute the cells to 2.5 X103~50X103/ml, depending on the growth rate of the cell line, and each microtitre plate can hold 20 ml of cell suspension. 4.
4. Transfer the cell suspension to a 9 cm dish and add 20 ul of cell suspension to each well in the middle 10 columns of a flat-bottomed 96-well plate (80 wells per plate) using a multichannel spiker, starting from column 2 and ending at column 11, adding 0.5X 103-10X103 cells per well. 5. Add 200ul of culture medium to the plate.
5. Add 200 ul of medium to 8 wells in column 1 and column 12. Column 1 is used as a blank control for the plate reader, and column 12 helps to maintain the humidity of column 11 and minimize the "edge effect". 6.
6. Place the plate in a plastic lunch box and incubate at 37°C in a humid environment for 1-3 d. Drugs can be added when the cells enter the exponential growth phase.
7. For non-adherent cells, prepare cell suspension with fresh medium, dilute the cells to 5X 103-100X103 cells per ml/m l, and inoculate only 100 ul of cell suspension into a round-bottomed 96-well plate, then add the drug immediately.
Addition of drug
8. A 5-fold serial dilution of the cytotoxic drug is made in the medium and 8 concentrations are prepared. Concentrations should be chosen so that most cells are killed at the highest concentration, but not at the lowest. A smaller range of concentrations can be used as long as the toxicity of the drug is known. In general, three plates should be used for each drug so that three parallel assays can be performed in a single experiment. 9.
9. For adherent cells, remove the medium from the wells in columns 2-11, which can be accomplished by attaching a hypodermic needle to the suction tube. 10.
10. Add 200^1 freshly prepared medium to 8 wells in columns 2 and 11, which serve as controls. 11.
11. Add cytotoxic drugs to the cells in columns 3-10. Only 4 wells are needed for each drug concentration, so that rows A-D can be used for the first drug and rows E ~ H for the second drug. 12.
12. Transfer the drug solution to a 5 cm dish and add 200 ul to each of the 4 wells in each group using a 4-channel spiker. 13. Place the plate back into the plastic dish.
13. Return the plates to the plastic box and incubate for the determined exposure time. For non-adherent cells, dilute the drug to two times the expected final concentration and add 100^1 to the wells containing 100^1 cells.
Growth Period
14. At the end of the drug exposure period, remove the medium from all wells containing cells and add 200 ul of fresh medium. If the cells are non-adherent, the plate is centrifuged (5 min, 200 g) to allow the cells to settle. Then remove the medium with a fine needle without disturbing the cell mass. 15.
15. Change the medium to 2-3 P D T daily.
Estimation of the number of surviving cells
16 . At the end of the growth period, 200 ul of fresh medium was added to each well, and 50 ul MTT was added to all wells in columns 1-11.
17. Wrap the plate with aluminum foil and incubate at 37°C for 4 h in a humid environment. Please note that 4 h is the minimum incubation time required, and can be extended to 8 h.
18... Discard medium and (centrifuge for non-adherent cells), add 200ul of DMSO to all wells in columns 1-11 to dissolve residual MTT-methylated crystals. 19.
19. Add glycine buffer (25 ul per well) to the wells containing DMSO. 20.
20. Record absorbance at 570 nm immediately, as the product is unstable. The plate reader is zeroed with the wells in column 1 containing medium and MTT but no cells.
MTT assay analysis
21. Plot the graph with drug concentration as the horizontal coordinate (X-axis) and absorbance values as the vertical coordinate (Y-axis). 22.
22. The average absorbance values obtained from the wells in columns 2 and 11 were used as control absorbance values, and the drug concentration required to halve the control absorbance value was the IC50 concentration. Occasionally this is not the case, indicating that the number of cells inoculated is not consistent throughout the plate.
23. Absolute absorbance values are obtained and plotted so that they can be compared with the control absorbance, and then the data are converted to a percentage inhibition curve (Figure 22.6) to normalize the series of curves.
