MTT assay is not working - (Oct/01/2009 )

Hi,

I have to do cytotoxicity tests. I use the MTT assay but it is not working and I really do not understand why. Here is the protocol that I follow:

- I seed cells into a 96wells plate.
- I let them 24h at 37C
- I add the drug (or water for control wells)
- I let them for further 24h at 37C
- I add MTT prepared in PBS so as MTT concentration in wells is 1mg/mL
- 4h of incubation at 37C
- I empty wells, I add DMSO

All wells are white, there is no absorbance. I tried to change several things:
- changing the MTT (in case it was to old)
- prepared it in unsupplemented medium
- not adding water in control wells (in case there was an osmolarity problem)
- changing the cell density (in case they were too confluent. I seeded 10,000 cells / well at the beginning, now I seed 3,000 cells/wells only)

But nothing worked.
In fact, when I check cells, they are looking ok after 24h, just before I add the drug. But after 48h, they are not looking ok and I think they are dying, EVEN in control wells where I didn't add the drug.

Do you know why the cells are dying like that ? What else could I try ?

Thanks,
Regards.

I think your problem is the fact you say I empty the wells, T

his is a common shortcut taken by many researchers doing the MTT or the Neutral red assya, the problem with turning over the plate onto a piece of tissue (which is what i pressume you do but correct me if i am wrong) is that it dislodges both cells and MTT crystals-even healthy living cells.

Therfore there is no MTT left to be solubilised by the DMSO,

Have you tried viewing the cells just before removing the MTT to see if you can see some purple/blue crystals forming within the wells, or just after removing before the PBS wash? if so, and you dont get any colour change then this loss of cells is most likely the problem.

If there are no crystals present at this time it could be something else but as you say the cells ar alive after 24 and 48 h they then should produce the colour change from yellow to purple

When performing again never turn over the plate to empty wells (at any satge of the process) always aspirate out each well individually with a pipette, it not only works every time but the std deviation is always better in my experience.

Thanks for your answer.
Aspirating out each well one by one is what I do... I don't turn over the plate.
There is no blue crystal after 4h of incubation with MTT so it is not really surprising if all wells remain white when I add DMSO.

Cells are not alive after 48h or they are dying. I am working with fibroblasts. After 24h they look ok, but after 48h they don't have the normal shape of fibroblasts.
Something happens during the 2nd day and causes the cells to die. But what ?

What concentration MTT do you make up, there are two ways to do the MTT assay, one is to incubate for 4 hours (the way you do it) using 0.5 mg/ml final conc another way is to incubate for 24 hours using 0.05 mg/ml maybe you have mixed up the lower concentration with the 4 hour incubation, i have attached my full protocol by the way we add the MTT in medium not in PBS for the 4 hours.

1. Cells were counted and seeded into a 96 well plate at a density of 0.5 x 10 4 cells per well (this works best for my cell type)

2. The plates were incubated at 37˚C and 5 % CO2 for 24 h to allow the cells to attach

3. 24 h later the culture media was removed and the cells were washed with 100 µl PBS

4. 100 µl of test chemical dissolved in dimethyl sulphoxide (DMSO) and culture medium was added to the cells, a negative control of culture medium only was included as was a control with culture medium containing 0.1 % DMSO (which was used as a solvent for the test chemicals)

5. The cells were incubated at 37°C for 24 h

6. 24 h later the test chemical was removed and the cells were washed with PBS

7. 100 µl of culture media containing 0.5 mg/ml MTT (Thiazoyl blue tetra-zolium bromide) was added to each well for 4 hours

8. After 4 hours incubation the media containing MTT was gently removed, washed gently in PBS and 100 µl DMSO was added to each well to solubilise the crystallised formazan product

9. After 10 – 15 min incubation at RT the wells were examined to ensure crystals had been fully solubilised and the plates were read on a Gen5 plate reader at 540 nm and a reference wavelength of 690 nm

10. The absorbance readings for 690 nm were subtracted from the 540 nm readings and the results were adjusted by dividing the average by the 0.1 % DMSO control to adjust for any toxicity that may have occurred in this control treatment set

I use a final concentration of 1mg/mL and incubate the plate for 4h.
I prepare MTT in PBS but I also tried to prepared it in medium or even in unsupplemented medium and the result was the same: no abscorbance at all...

I directly add 100µl of drug to the 100µl of medium added the first day. So I don't wash cells with PBS. On the 3rd day, I add 25µl of MTT (stock solution: 9mg/mL) into 200µl (drug+medium) so it gives a concentration of 1mg/mL.
Do you think that not washing cells could explain why it is not working ?

Perhaps there isn't any cytotoxicity? Do you have a positive control for cell death?

Also note that the limits of the MTT assay mean that you need about 10,000 cells per well for it to be detected.

If there was any cytotoxicity, all cells would be alive and wells would become purple. And that is not the case.
What do you call a positive control ? The only control that I have is wells where I do not add the drug. I just let the cells grow. But these cells are alive after 24h, but not after 48h. That's why I finally used 3,000 cells / wells only: I thought that if they were dying, it was because they were too confluent, too many.

I have only used 5000 cells per well as it was optimised to be the best concentration for these cells however coleagues of mine use Hep G2 CaCo-2 and other more widely used cells and have seeded at up to 50,000 cells per well.

your right the positive control would be something that would kill all the cells and give no colour change, we use 1 % triton X but anything that will kill all the cells could be used 1 - 10 % DMSO, 1% SDS etc.

However this is not the problem with your assay, the problem is the cells are all dead or at least not giving colour change

As the negative control wells with no drug added to it also start to die after 48 hours, This should not happen- would they die after 48 h culture in a flask when not doing the assay?, i doubt it, so maybe they are missing something during the assay that they usually have in the culture media when culturing them i.e. growth factors etc.

Or maybe the 96 well plates you are using are not the same type/make as the flask and they are not attaching properly to it (i.e. cell plus -collagen coated flask versus non cell plus plates)

Also If you cant see a colour change in cells when you know they are alive the problem could be with the MTT itself so i would recommend buying in a new bottle and never using stock after 3 - 4 weeks (i know you say you never do this anyway)

Apart from this i really dont know what it could be, but maybe try get your hands on a second type of cell line from your lab and try the assay using all previously made stocks if it works then it could be your cells if it doesnt its proably the reagents.

Cotchy.

I just re-read your response i note you dont remove your drug when adding the MTT, maybe there are some interactions between that stops the MTT working

i think you should remove all drug/media when 24 h incubation is over (it alkso means you have a 28 h incubation instaed of 24 h) wash then ad fsh media with MTT for 4 h to see if you get a reponse

Even if you dont have a test set up today maybe try it for 4 h today by adding the fresh media/MTT to healthy cells in a culture flask at the right concentration and se if yoy can notice the formazan product appearing

Cotchy.

slyphe on Oct 2 2009, 08:06 AM said:

What medium do you use?
Fibroblasts usually require FBS:
I use 10% in normal growth culture and 2% in 96 well assays.
If you like to do a test within 24-30 hours, I recommend you perform a preliminary test with increasing cell concentrations from 3 000 to 10 000 cells per well to find the right cell density that works for your assay.
I also use the MTS assay CellTiter Aqueous One solution (Promega): You change to fresh medium (100 ul per well) and add 20 ul of the reagent. That's it! No further steps required! You can choose an incubation time between 1-4 hours (I personally use 2 hours) before the read-out of the plate.

Good luck