MTT

[Dr Engelbert Buxbaum]

Am 01.02.2008, 17:09 Uhr, schrieb Tom Anderson <ucgatan from ucl.ac.uk>:


> I think if i had access
> to a Coulter counter, i'd be using that instead, and getting direct
> measurements of cell number and volume.

The problem is that a Coulter counter does not distinguish between dead  
and alive cells. Also, it tends to be more work. And plate readers are  
much more wide spread than Coulter counters.

>> 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?
>
> You need a standard every time you do the experiment. I'm not sure if you
> need a whole standard curve, or just a single control well, where you
> haven't added a test compound. The latter should be fine if the assay's
> linear. Maybe three control wells, to be sure.

The usual way is to do a concentration dependency: Suspend your cells in  
20 ml medium and pipette 0.2 ml aliquots into each well (taking care that  
the cells can not settle). Allow cells to attach o/n, remove the old  
medium and replace with 150 ul fresh. In the wells numbered H, add 150 ul  
stock solution of the chemicals you want to test, mix gently. Take 150 ul  
to well G, mix gently and take 150 ul to well F. Continue until you have  
reached well B, where you discard the excess 150 ul. With this procedure  
you get a series of different concentrations of your chemicals, doubling  
 from well to well, except in well A where the concentration is 0. Let the  
cells grow until they approach confluency in the well A, then perform your  
assay. Plot the absorbance you get against the the concentration of your  
chemical, you should get a S-shaped curve starting at 100% growth in the  
first wells. The relevant parameter is the concentration at which you have  
only 50% growth, this is called IC50 and obtained by non-linear curve  
fitting. These IC50 values you can use to compare the toxicity of  
different compounds on the same cell type, the same compound on different  
cell types, or the same compounds on the same cells under different  
conditions.

It is best to do the assays in triplicates, so you can test 4 different  
chemicals in each plate.