Jose de las Heras
(by josenet from tiscali.co.uk)
Fri Feb 1 16:33:56 EST 2008
"Yvonne Couch" <yvonne.couch from dpag.ox.ac.uk> wrote in message
news:mailman.441.1201825059.2451.methods from net.bio.net...
>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?
> My next query is that after running my standard curve I'm supposed to pick
> 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?
> These seem like fairly obvious questions but I am genuinely confused!
Well, I've never done one of these tests, but I have reasonable experience
quantitating signals (southern, RT, microarrays, etc).
The reason to try various amounts of cells is to find a range in which you
are confident you can detect differences in... in this case mitochondrial
activity. Any system such as this has a useful range. Below a particular
threshold, the signal is too low. Above a certain threshold the system is
saturated and you don't get a stronger signal when the activity is higher.
If you plate different amounts of cells (known) and plot the signal you
measure vs. the number of cells, the useful range is found where the
relationship is linear. After a certain point, the graph will level... if
you use a number of cells in the linear part of teh graph, you can then
quantify the difference in mitochondrial activity.
Does this make sense to you?
(by the way, not a stupid question at all...)
This is related to a petty hate of mine... when you see on a paper a Western
blot, for instance, and the show you the pattern corrresponfing to whatever
antibody, and below some other antibody (such as tubulin, or whatever) as a
"loading control" to show that all lanes contain similar amounts of
protein... and many are saturated! Well, if you saturate, sure, they will
all look the same! Watch out for it, it's *very* common. Usually it doesn't
destroy the point the paper makes as they have other evidence, but still...
More information about the Methods