below 10%..., Review article of your compound to fix your DMSO %  and then use various concentration of your compound to do MTT Assay.  what is  your compound name?

In my experiences with muscle cells I used 0,1% (someone used 0,5%, the max is 1%), it is not only a problem of toxicity but also to understand if DMSO and not your compound caused changes in your cells responses. As asked before what is your compound ? Do you have to dissolve your compound only in DMSO? Do you have articles about your compound concentrations used in culture cells? You can find the commonly used compound concentrations and than decide the DMSO concentration itself.

I totally agree with last 2 answers. Final conc for adding a compound to cells in culture should be at or below 1%. Fopr most mammalian cells in my experience 10% would kill them or at least potentially induce abnormal genotypic or phenotypic affects so if in doubt stick to 1% or below !!

No more than 0.1%, if possible, And always add controls with DMSO and without your compound, to check for the effects of DMSO.

I agree with Lilian

I absolutely agree with the above suggestions on less then 1% DMSO, personally I would use 0.1-0.5%. When we use DMSO as a free radicle scavenger we started to see adverse affects to control cells at concentrations between 0.8-1%. 

Thank you very much for all of your valuable comments. this is not a known compound. and not soluble in water. that is why i tried to dissolve it in DMSO. Once again highly appreciate your valuable comments.

Hello Again

upto 1% DMSO is generally considered the first solvent of choice over and above water. You should therefore alway try dissolving in DMSO first and NOT water. I participated in number of drug discovery projects in prior positions and we would always use neat DMSO and try and ensure the final working concentration of DMSO (plus compound) in working assays was 1%. Above that DMSO can have deleterious effects on cells in tissue culture and above 5% DMSO can start to denature proteins by virtue of melting hydrogen bonds. In general most compounds are more soluble and retain activity better in DMSO than water explaining my prior point. Somecompounds however ppt in DMSO and in those situations mixtures of DMSO and ethanol tend to work well. It comes down to miscibility of compounds and retaining structure/activity. Many compounds and indeed biological molecules lose activity in nascent water which is why biological experiments are conducted in isotonic solutions like PBS and not simple water. The same rational can apply to compound used in drug discovery experiments. Enough said !! Good luck

Hello Laurence,

Thanks a lot for the advice. yes I dissolved my compound in 100% DMSO and then diluted up to 0.2%. It will work I guess. 

Cheers

If your compound has miscibility/soulubility issues in neat DMSO try mixtues of ethanol/DMSO. Avoid water if you can  !!

Sometimes the toxicity of the compound also matters. I've seen toxicity with as low as 0.1%. In this case you have to run a visibility study over 72 hrs by evaluating the viability every 24 hrs and observing the changes. A compound may have 70-85%  viability at 24 hrs but drops below 50% by 72hrs.

to be very sure and to avoid all possible interference with the result, I would never cross 0.1% DMSO. if possible increase drug concentration. that would be 0.5ul/well for 24 well plate assuming that you use 500ul final volume.

As a rule of thumb DMSO shouldn't be more than 0.5% in your cell culture. some cells can tolerate well up to 0.7-0.8% other are sensitive even at

As per my knowledge, DMSO concentration shouldnt exceed 0.1%, if you have doubt with that add equal amount of DMSO in differing drug concentrations. So you ll get equal amount of effect of dmso............

Hi everyone,

Though my response to the question might be late but can still be valid. i will advise a dose response studies be conducted to determine the maximum tolerance level to DMSO. This varies within cells. However, most studies recommend 0.1 to 1% DMSO.

I always use the final DMSO concentration of

Very confusing too. All are different answers. Logically, we usually dissolved compound in 100% DMSO and make sure the compound is dissolved properly. Then we have to use this compound to treat cells. For MTT assays, we normally do 2-fold serial dilution in the 96-well plate directly. First well, media with cells (200 ul) and compound is 2 ul to add in. So if we calculate this, DMSO is 1%. Then next well has 100 ul cell+media then we take 100 ul from 1st well and added into next well. So next well were become 0.01% . Like-wise, DMSO % will be reduced 100 times for next wells. We included cells + 1% DMSO final concentration well too. So here, we observed that even with 1% DMSO, cells are still OK, not effecting any. I haven't test yet with more than 1%.

1 % is more than too much. I generally use a dilution factor of 1:5000. That means 0.25 uL DMSO containing dye or drugs in 1.5-2 ml of media.

I think people who believes that 1% DMSO is 'OK' for cells they might not have checked the status of the mitochondria. Functionality of the mitochondria gets perturbed significantly. Hence, from my practical experience I would suggest to use 1:5000 fold dilution.

I always try to maintain 0.5% maximum final DMSO % in a cell culture plate.

0.1~0.2% for maximum.

I have seen that the cell membrane becomes more permeable and more cells start dying if % DMSO is more than 0.5 % by volume in cell culture media. I saw that the phagocyte cells like RAW 264.7 engulf (it might just be more internalization as well) about 10 to 20 % more in a media with about 0.46 % DMSO as compared control cells in regular DMEM media. See supporting information figure S7 in paper " Joshi, Bishnu P., et al. "Surface-Modified Macrophages Facilitate Tracking of Breast Cancer-Immune Interactions." ACS chemical biology (2018). "