MTT assay

Hello, i am Stanley and this is my second blog posting.

The MTT assay is a quantitative colorimetric method used to determine cell proliferation. It utilizes the yellow tetrazolium salt which is metabolized by mitochondrial succinic dehydrognase activity of proliferating cells to yield a purple formazan product. This purple formazan is then solubilized and the concentration is determined by reading under an optical desity which ranges from 500nm to 600nm. As the MTT assay only detects living cells, it can be use to measure cell cytotoxicity, proliferation or even activation.

Part of my experiment will include the use of MTT assay to determine both the growth curve of both normal lung cell (MRC-5) and large lung carcinoma cells (H460) which are seeded in a 96 well flat bottom plate. The advantages of using MTT will include high accuracy and the exclusion of using radioisotope.

Below are steps for performing the MTT proliferation assay:

Preparation of MTT stock

1) Adding 1ml of sterile PBS into the 5mg vial of MTT

2) Votex the mixture till all of the MTT is dissolved (takes approximately 3 to 5mins to dissolve completely)

(Note: the MTT stock can be stored for four weeks at 4 degrees celsius protected from light)

Labeling of cells


(Before adding the MTT stock)


3) Add 10µL of MTT stock to each well ( The wells will turn to a slight yellow in colour)

4) Incubate at 37 Degrees Celsius and 5% carbon dioxide concentration for 4 hours (1st incubtion)

5) After incubating the plates for 4 hours, remove all but 25 µL medium from each well

6) Add 50µL of DMSO into each plates

7) Incubate the plate for the second time at 37 Degrees Celsius and 5% carbon dioxide concentration for 10mins (2nd incubation)


(After the second incubation)


Absorbance reading

8) Read the absorbance at 540nm using a microplate absorbance reader

The above steps is a quick alternative to the conventional steps which uses SDS sodium dodecyl sulfate - HCL solution instead of DMSO to solubilize or dissolve the purple formazan. However, the conventional steps will require an addition of 4 to 18 hours of incubation time ontop of the first incubation stated in step 4 and has to be read at 570nm instead of 540nm.

Some important points to note are that:

1) DMSO is a powerful solvent and is able to rapidly penetrate the skin and it is important to avoid skin contact with DMSO

2) Both DMSO and the MTT stock are sensitive to light and must be kept away from light to prevent degenerative effects or inaccuracy

Thank You