Re:MTT assay법에 대해 아시남요??----여러가지 MTT assay 올립니다. 참고하세요

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The measurement of radiation survival using a clonogenic assay, the established standard, can be difficult and time consuming. The MTT assay, based on the reduction of a tetrazolium salt to purple formazan precipitate by living cells, as a substitution for clonogenic assay was assayed to define the optimal condition for performing this assay in determination of radiation sensitivity.

Four human cancer cell lines - PCI-1, SNU-1066, NCI-H630 and RKO cells have been used. For each cell line, a clonogenic assay and a MTT assay using Premix WST-1 solution, which is one of the tetrazolium salts and does not require washing or solubilization of the precipitate were carried out after irradiation of 0, 2, 4, 6, 8, 10 Gy. For clonogenic assay, cells in 25 cm2 flasks were irradiated after overnight incubation and the resultant colonies containing more than 50 cells were scored after culturing the cells for 10-14 days. For MTT assay, the relationship between absorbance and cell number, optimal seeding cell number, and optimal timing of assay was determined. Then, MTT assay was performed when the irradiated cells had regained exponential growth or when the non-irradiated cells had undergone four or more doubling times.

There was minimal variation in the values gained from these two methods with the standard deviation generally less than 5%, and there were no statistically significant differences between two methods according to t-test in low radiation dose (below 6 Gy). The regression analyses showed high linear correlation with the R2 value of 0.975 - 0.992 between data from the two different methods.

The optimal cell numbers for MTT assay were found to be dependent on plating efficiency of used cell line. Less than 300 cells/well were appropriate for cells with high plating efficiency (more than 30%). For cells with low plating efficiency (less than 30%), 500 cells/well or more were appropriate for assay. The optimal time for MTT assay was after 6 doubling times for the results compatible with those of clonogenic assay, at least after 4 doubling times was required for valid results. In consideration of practical limits of assay (12 days, in this study) cells with doubling time more than 3 days were inappropriate for application.

Thus MTT assay offered a rapid, simple method for the assessment of radiation sensitivity of cell lines. However, the necessity to optimize the conditions of the colorimetric assay for each cell line may limit its usefulness in determining radiation sensitivity.







MTT Assay

This procedure is for cells in 96 well plates, if larger plates are used then adjust volumes accordingly.

1 Make a solution of 5mg/ml MTT dissolved in PBS and filter sterilise.

5 hours before the end of the incubation add 20m l of MTT solution from step one to each well containing cells.

Incubate the plate at 37튏 for 5 hours.

Remove media with needle and syringe.

Add 200m l of DMSO to each well and pipette up and down to dissolve crystals.

Put plate into the 37튏 incubator for 5 minutes to dissolve air bubbles.

Transfer to plate reader and measure absorbance at 550nm.






MTT assay

◎ Current method
Required materials
MTT : 2mg/ml MTT in PBS DMSO

Brief protocol
1. Plate cells (104-106 cells)in 96-well (flat form).
2. Incubate for appropriate time.
3. Add 25µl of MTT soln. to each well.
4. Incubate for further 4hr.
5. Decant gently supernatant.
6. Add 100µl of DMSO, and resuspend the pellet for 30 min during shaking.
7. Measure OD in 540nm.




MTT Assay

The MTT assay is a quantitative colorimetric assay for cell survival and proliferation, based on the ability of live cells to utilize a pale yellow substrate (a tetrazolium salt) and its subsequent modification into a dark blue formazan product. The tetrazolium is cleaved in mitochondria, so the reaction only occurs within living cells. The assay detects living, but not dead cells, and the signal generated is dependent upon the degree of activation of the cells.


Materials: Stock solution of Thiazolyl Blue (MTT), 2.5mg/ml in deionized H2O (store at 4oC)
0.04 M HCl in isopropanol ( store at RT)


Method:
1)For every 500ul of media in a well, add 20ul of MTT solution.

2)Place the plate back into a 37oC incubator for 3-4 hrs.

3)After the allotted time, remove the media by pouring into a sink (Do not aspirate the media from the wells).

4)Place 200ul of isopropanol into each well and place on an orbital shaker for several minutes to dissolve the precipitate.

5)Take 90ul of isopropanol solution and place it into a well on a 96 well plate. Do this twice for each sample.

6)Read the plate at 590nm absorbance.