MTT vs.3H Thymidine
gautam at mcbl.iisc.ernet.in
Wed Aug 4 23:07:56 EST 1999
A little addition of what was mentioned about mitochondrial function
during cells undergoing apoptosis.
The number of mitochondria and the functional capacity varies between
cancerous and non-cancerous cells. Also between non-transformed yet
immortal cell lines. Hence once has to be really careful in ventruing to
do an MTT and base their discussions on such results.
Moreover, if you want to correlate your data with thymdine incorporation,
mitochondrial activity and the MTT assay you have done its very simple but
a little time consuming, but the results you get will be 100% correct at
the end. Take as follows :
1) a single cell culture. Now from this, take
1a) an aliquot for MTT assay
1b) an aliquot for thymidine incorporation
1c) an aliquot for fluoroscence microscopy studies for mitochondrial
status - whether functional or non functional, which will also give you
the approx picture of the density of mitochondria per cell.
1d) morphological data of cells undergoing apoptosis
1e) an aliquot for trypan blue.
All when put together will give you the complete picture of what is
happening in one single cell type/system with your experiment. Especially
if you are studying apoptosis.
best of luck.
On Wed, 4 Aug 1999, Frederick Coffman wrote:
> If you are interested in measuring DNA synthesis, incorporation of thymidine
> is a direct measurement of that property. The MTT assay is more an assay of
> living vs. dead cells; more specifically it is an assay of cells with
> functional mitochondria. The yellow MTT substrate diffuses into the cells
> and is reduced to a purple formazan product by mitochondrial dehydrogenases,
> thus the amount of purple color in a well (of a 96 well plate) is directly
> proportional to the number of living cells with functioning mitochondria.
> There are some caveats - cells undergoing apoptosis can show reduced
> mitochondrial function hours before they would be classified as dead using
> other assays, and in some conditions (like giving very low concentrations of
> TNF to some tumor cell lines) cells show enhanced mitochondrial function wrt
> this assay, but in general the MTT assay is a fairly reliable way to measure
> cell death.
> But if you're interested in DNA synthesis, by all means measure it directly.
> In article <37a5be05.2333583 at news.mcgill.ca>, skim7 at po-box.mcgill.ca
> (Pianoman) wrote:
> >Hello Experts,
> >In our lab, there is a little debate on the method and I wish anyone
> >can give us idea.
> >We are testing the effect of some reagents on cell proliferation and
> >DNA synthesis. We did some cell counting experiment using
> >hemocytometer. We are interested in DNA synthesis and if possible,
> >ribonucleotide reductase activity.
> >At the point we did the experiment using hemocytometer, what is better
> >method between MTT and 3H thymidine uptake? Which one can we draw
> >more conclusion out of data with?
> >I would really appreciate any kind of information.
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