MTT assay + suspension cells - (Oct/09/2007 )

Hello, anyone has any experience doing MTTs in suspension cells? What is your protocol?

I'm my lab most people work with adherent cells and use DMSO to solubilise the MTT formazan end product. We "inherited" an old method in which we after adding the MTT substrate + incubating for 4 hours, we add 10% SDS / 0.01M HCl.

I did a batch of MTT experiments early this year and all of them worked as expected. However, this last summer, a colleague of mine tried doing some MTTs with the very same cell line I use. Basically what she observed was that the formazan is successfully produced (since you can see the blue crystals under the microscope), but the SDS/Hcl seemed to fail to sollubilise the solution. We got like a blob of blue aggregate, but not uniform colour in the wells.

I repeated the MTT's I did early in 2007 but got the same kind of blue smear. Also did some controls a few days later and the MTT worked. Upon repetition, it didn't work. So we get this kind of irreproducibility now when there was good results a few months ago.

We ordered new MTT reagents and made a new batch of SDS/HCl but obtained the same results.

Any ideas? Similar protocols for suspension cells would be much appreciated...

Since MTT creates an insoluble product (formazen), can't you just spin it down purple formazen and then resuspend in 100% isopropanol? Thats what we normally do for adherent cells anyways, but it should work for cell suspensions also (given you have enough cells).

If for some reason you can't do that, I would try to use Wst-1 from Roche (even though it is pricey, it gives tighter error bars). Wst-1 is nice because you just add it to you cells and then read it on the spectrophotometer, since the end product is a soluable formazen.

Hope this helps...

How long do you incubate SDS in cells?
Where do you keep SDS? Fridge?
How old was the MTT solution?

We incubate SDS solution for 30 minutes after the 4h incubation with MTT.
We keep the SDS solution in incubator(I know this isn't very "hygenic") and MTT solution in PBS is prepared at the moment of the experiment.

I'll look up the exact concentration of MTT and composition of SDS solution and let you know ASAP.