Did you remove culture medium before the adding DMSO? To avoid formation of bubbles, use gentle stirring on plate shaker. Then, have a little patience.

Thanks a lot! Yes, I removed the medium (leaving about 25 ul in the well), and then added 50 ul of DMSO pipetting esch well.

Hey,

after having tried many solvents, I also found DMSO to be the best choice. If necessary, you can significantly reduce variances by adding ammonia (cf. www.sciencedirect.com/science/article/pii/S0003269711007093).

Thank you for your suggestion! I have downloaded the article and I will try.

We followed the method of Mosmann (1983). We added Acid-isopropanol (100

uL of 0.04 N HCI in isopropanol) to the wells after removing media and washing with PBS

You can try adding a lysis buffer, which we use in our lab, and it is added direclty to the medium (I use this only with suspension cell cultures or neurosphere cultures, but I don't see a reason why it wouldn't work with adherent cells). The buffer is:

10 g SDS in 100 ml H2O + 83 ul 37% HCl (mixed under the fume hood)

from this buffer we add 100 ul per well for the 96-well plate format (without removing the medium from the cells), and for me it works perfectly.

After removing the MTT medium, formazan crystals precipitated on the bottom of the well are effectively dissolved using 100uL DMSO per well.

The best option is to remove the entire medium and rinsing twice in PBS to avoid the background noise, before You add MTT solution. Carefully remove the MTT and dissolve cristals in DMSO. Then, In order to get rid of bubbles I would recommend short centrifugation.

Either isopropanol or DMSO - both work equally well. Avoid the use of pipetting for dissolving the crystals as this is causing the bubbles. You can use an orbital shaker for as long as it takes to fully dissolve the crystals.

I do not remove the culture medium. I just added 100 uL solubilizing solution (10% SDS in 0.01 M HCl) to each well and incubated overnight in a humidified 5% CO2 atmosphere. you can try that you will not see bubbles.

Supernatants were removed and the formazan crystals were solubilized in DMSO (100 µl/well) for 30 minutes at room temperature

The best solvent for formazan crystals is DMSO which you used, But there is no need for pipetting to be solubilize. I usually leave the 96 plate 10 minute in incubator for solubelization of formazan crystal in DMSO. I hope it works for you too.

Hi, I agree with Anna Linge.

I am always using isopropanol. And it cheaper then DMSO ;-).

Try that.

As you know the number of cells is an important parameter in MTT assay.it is neccessary to obtain better result in OD and better solubility in DMSO,reduce the number of cell before doing MTT assay.Be successful.

Thank you all very much for your valuable help!

you can try mixing DMSO in a separate vial and once it gets dissolved take the purple solution in 96 well microtiter plates for reading. this way you can avoid interference of bubbles. repeated pipetting may also result in bubble formation.

Two techniques are described: using acidified isopropanol (original MTT protocol by Mosman et al) and the more convenient method using SDS (modified version by Tada). I used both but found the method by Tada much more convenient because it´s not smelly, dissolving the crystals worked well without much effort (37°C incubator o/n, shaker if you have, but works fine without) if the incubation time with MTT is tuned well (if your cells are very active you can decrease the incubation time to 1h - maybe less if you have many and highly active cells). An additional plus with Tada´s method is, that cells don´t need to be washed so it´s good for testing many conditions/samples  etc on 96-well plates. Find the method by Tada attached, including the reference for citing. The method by Mosman will follow in the next post as it seems that I can't attach two files...

and here is the protocol for the original Mosman method...

One more thing to note: MTT assay is a bit tricky for analysing cell proliferation. It measures also cell activity (NAD(P)H-dependent oxidoreductases). If you have the same number of cells, one in a resting stage the other metabolically active, the metabolically active cells will give you a higher MTT activity. If you use immune cells (e.g. from the spleen), you should take this into consideration. Especially if your cells are treated with something that might activate them; comparing these cells to naive control cells (not activated) will give you the false impression that your cells proliferated. If you have no other option for assessing cell proliferation, I would recommend to actually go through the pain of counting cells manually to validate MTT.results. Of course you can start out with MTT to screen many conditions and limit manual counting to the relevant ones you identify by screening.

Though we are using the MTT assay for toxicity assessment, I agree with Christoph's comment about cellular metabolism. Especially before actually dying, cells exhibit a seemingly higher vitality ('hormesis effect'). If that proves to be a problem in your proliferation assay, you might try another test, e.g. resazurin or neutral red retention.

Try doing a calibration curve first of abs versus time DMSO is left in the well to solubilise the formazan. When the abs reaches a plateau you will have a definitive time needed for all your samples in the future (i.e. how long you will need to leave the formazan in your wells to fully dissolve the crystals).

In terms of solvent DMSO, Isopropanol or a mixture of HCL and Isopropanol are all popular.

After MTT incubation you have to centrifuge 10 min at 1000 rpm, then remove the MTT and added the DMSO.

The MTT assay seems very simple and attractive, but is not assigned to estimate proliferation, as Christoph reasonably noted above. Although some correlation will be seen, unlikely you will be satisfied by results. Activated splenocytes transform MTT weakly and debris from dead cells will negatively affect the quality of analysis. To estimate cell proliferation it would be more correct to use determining of 3H-thymidine incorporation or CFSE staining with subsequent analysis by FACS.

Hi all,

I have been doing Mtt assay, I have got purple crystals that solubilize in isopropanol easily. the problem is after removal of mtt and according to the standard protocol, I have to wash 2x with PBS, these crystals some times were removed with PBS after aspiration. is it normal? or, should I avoid washing after removing mtt.  I guess that  the formation of purple crystals that accumulate outside, results from the mitochondrial enzymes released from damaged cells which responsible for the metabolism of mtt outside the cells., and they should be washed out.     

am I correct?

Hi,

Very informative discussion.I have few questions as i read through different blogs and got confused.

1).MTT working solution must be 5mg/ml not 0.5mg/ml ?

2).What concentration of DMSO i must use to solublize the fromazen crystals ?

Thanks

Ammy

Hi Ammy,

I think 5 mg/ml is a very high concentration. We are using 1 mg/ml, but I think 0.5 would suffice too. You have to use pure DMSO (100%).

@ Ismael Obaidi (and others with similar considerations): there is usually no need to wash cells after MTT-treatment. After the incubation time you can just add the lysis buffer. Please read the original articles - they are full of information and would make many discussions obsolete if read :-) e.g. phenol red as a pH-indicator is usually no problem as the lysis buffer (acidic) will take care of this.

@iA Ia - Dear Ammy, 5 mg/mL is the concentration of the stock solution (it´s not the final concentration your cells are exposed to) according to Mosman or Tada protocols and works fine. Can´t comment on DMSO for solubilisation - SDS worked excellent for me (Tada et al protocol). Also keep in mind: solubilisation buffer should be acidic to take care of phenol red in case it´s in your growth medium.

Thanks Christoph.

We are using a solution of isopraponol,triton-x and HCL and its working prfctly for us

• Please could u explain to me the procedure you’ve done for MTT assay ? @kunal pal

Christoph Metzendorf how much the final concentration of MTT usually used? I works on dendritic cell.