(by yvonne.couch from dpag.ox.ac.uk)
Thu Jan 31 11:59:53 EST 2008
I have another (relatively stupid) question. This is definitely not my
education but my own brain not being able to understand the problem. I've
just started using the MTT assay to look at the toxicity of two different
compounds. I get how the assay works, mitochondrial reduction of the salt
to formazan which forms purple crystals, more crystals = more mitochondrial
activity. But I apparently need to run a standard curve with increasing
cell numbers in each well. I'm not really sure why. I've run it and I get
more purple as my cell number increases but surely this is just because
there are MORE cells and therefore the very nice line graph I have doesn't
really tell me anything except that more cells make more crystals? The
proliferation rate of the cells, no matter what their number, should
essentially be the same?
My next query is that after running my standard curve I'm supposed to pick a
cell number and use that in all the assays. I have picked 10k cells per
well since it was nicely in the middle of my graph but on different days it
has not given the same reading when untreated (0.3 absorbance one day 0.6
the next) so if I treat the cells with a compound how am I supposed to know
if it's doing anything to their rate of proliferation?
These seem like fairly obvious questions but I am genuinely confused!
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