MTT in just PBS or PBS and medium? - (Sep/06/2013 )

Hi

I've been doing some MTT assays for the last couple of months, following my lab's protocol.

Then I started searching for other protocols to write a more complete one for myself and I found a difference. I would like your advice on it.

In my lab's protocol we do like this:

- culture cells and treat then as determined

- remove media of every well

- add 10 uL of 5 mg/mL MTT solution (in PBS) to each well

- incubate for 3 hours at 37°C 

- remove MTT and add 100 uL of acidified isopropanol

- read at 570nm

The protocol works just fine, but in most other protocols I found that MTT solution is added TO THE MEDIA, and not TO THE CELLS as we do.

Does everyone know if this make much difference at the end result?

Thanks!

Yes, most protocols suggest that MTT solution is added to cells in medium. Where did you get your protocol? 

After adding MTT solution, you wish to keep viable cells alive during the incubation time. Since you have to incubate your cells for 3 hours after removing the medium and adding only 10 ul of MTT solution, I am concerned that how your cells can survive in 10 ul solution for 3 hours. 

That is the problem! I can't find the reference to the protocol we have been performing at the lab... People says "we always did like this"

I had the same concern about keeping the cells alive... But the formazan crystals are always formed and I get good reproductibility.

Now I am having doubts if I should keep doing this way or change it...

In my lab

I remove media before adding MTT

I think that PBS may keep cell living in short term.