(by pow.joshi from gmail.com)
Thu Jan 31 19:42:22 EST 2008
On 31/01/2008, Yvonne Couch <yvonne.couch from dpag.ox.ac.uk> wrote:
> 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
> 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
> 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?
Frankly, I have done a couple of MTT assays a rather long time ago, and so
my knowledge could be a bit jaded... however, here's what I think:
you would have to do a cell number standard curve, precisey to determine the
cell number where the assay is where the curve is not quite saturated. You
will get a graph that is a stright line upto a certain cell number after
which it plateaus out .... (perhaps, you could also check the cell density
in your well, and see if the plateau correlates with the confluence, if
you're using attached cells).
Further, I am not sure if you are going to caliberate you results as
arbitrary "absorbance" units or number of cells proportional to the
formazan? (for the latter, you would have to run a caliberation/ standard
curve for each experiment)
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
> 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
> if it's doing anything to their rate of proliferation?
well, I imagine you will have varying results since you are using a "living"
biological system. This would mean that on some days your cells are happy,
on others sad/ stressed/ partyed and zonked. Sometimes the errors in
pipetting and counting that also add up...perhaps you're having a bad day :)
You would have to have a untreated control or run a standard curve each
day, which will give you the baseline reading, that you may want to subtract
from the experimental set.
Hope that helps,
These seem like fairly obvious questions but I am genuinely confused!
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