My project is based on wound healing. Can I use the MTT assay for proving cell proliferation? If not what should be the alternative assay I should perform?
yes Mtt assay can be used to,assess cell proliferation in vitro culture
XTT is also an excellent way for measuring proliferation (derivative of MTT)
in my opinion, test MTT should be mainly used to determine the cell viability and also the IC(50) value.
yes, I ever do it, using time course. The principle of MTT or WST is measuring the viable cell and to measure the IC50. That is better if you try using tryphan blue, you can measure the dead cell and live cells.
WST : http://indonesianjpharm.farmasi.ugm.ac.id/index.php/3/article/view/13
MTT : http://www.arjournals.org/index.php/ijpm/article/view/363
I guess that MTT is only a rough measurement of cell proliferation, when looking under a light microscope the distribution of blue colour in cell culture experiments , I noted that some cells remains yellow whereas other appears heavily blue. So spectroscopic determination only gives an average value of MTT reduction and cell activity. I would prefer DNA determination to follow more precisely the evolution of cell mass.
jeah, in theory you can use mtt assays to measure cell proliferation. anyhow its a very blurry assay. to get more clear results i would recommend a BrdU or a CSFE dye staining.
Although you can use MTT assay as a rough measure for cellular proliferation, it is not a standard method of cell proliferation estimation. MTT color development is just based on overall viable cells available for the reaction. MTT results are the sum obtained from cellular proliferation and cell death. You can use Ki67 immunostaining or BrdU staining for accurate measurement of cellular proliferation. Both of these can be used under in vivo or in vitro conditions.
Many papers have been published using MTT as a surrogate for cell number and hence, cell proliferation. However, as with all surrogates, the validity of one's conclusions depend on the validity of the assumptions underlying the use of the surrogate. The use of MTT is only appropriate in the region of linearity between counted cell number and MTT signal (density or colorimetry) values.
What you really want to have measured is the number of cells present as a function of time and use that to produce a net growth (or cell loss) curve. The roles of proliferation and cell loss then need to be assessed as well to understand what is happening.
MTT was used as a quick (and dirty) way to estimated cell numbers from densitometry or colorimetry scanner data. We now have much more sophisticated cell imaging (and counting) methods and they should be used instead, where feasible. There are many morphometric programs that will recognize individual cells in a well or colony and quantitate their numbers as a function of time. Much more reliable data are obtained this way.
Using MTT assay, you can not determine the cell count, but only the cell viability
Using the density of MTT staining, many labs have attempted to take this as a surrogate for cell numbers or density in a plate or well. There was a decent linear correlation between cell number and stain intensity (density) that was used to justify the surrogacy. But the range of linearity was limited. The cell count estimate was based on cell counts and MTT image densities.
Dear Viji,
I read above replies and I am totally agree that MTT can give you a rough estimation of cell proliferation but using MTT as a cell proliferation assay can give you false results also, because it doesn't give you exactly the MTT reduced by cells over a curse of time but only within a narrow time window.
I have observed the same in my experiments where cell proliferation by MTT WAS giving very unexpected results and when I measured the cell proliferation by a standard proliferation kit (EZBLue), then the results were totally contradictory to MTT but were in accordance to the expectation as EZBlue give you a more real picture of cell proliferation over 8-24 hrs. of culture so more reliable than MTT. Other good alternatives are BrDU incorporation assay.
So I would like to suggest "DO NOT USE" MTT as a measure of cell proliferation as it can create problems for you in future and then you might need to repeat your experiments with some standard cell proliferation assay. So I will advice to use some standard method as described above for the same.
Hope it helps !
Hi Viji,
We use the MTT assay a lot in our lab, one of the main problems with this assay is that it doesn't distinguish between anti-proliferative and cytotoxic effects as it only allows you to see changes in cell number over a short window of time. If you have a flow cytometer available I would suggest using the Oregon green assay to investigate changes in cellular proliferation. Oregon green is a cell permeable dye which once inside the cell is processed by cellular esterases causing it to become fluorescent and bind proteins within the cell. Every time the cell divides, the fluorescence is halved so with each division there is an exponential decrease in fluorescence, which allows you to monitor the number of replications the cell goes through by flow cytometry. I have a protocol I can send you if you are interested =)
Christopher, you said: There are many morphometric programs that will recognize individual cells in a well or colony and quantitate their numbers as a function of time. I appreciale if you could give me more information abour this programs.
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