Insoluble drug protocol?

Model Organism: Worms Methods & Reagents
1

Hi all, I could really use some help with C. elegans drug treatment. I’m wondering if anyone has had a water-insoluble drug that they have had success with and how you used it.
I am trying to use a water-insoluble drug, but it doesn’t seem to be very effective. When I put the drug in liquid, it precipitates.
Some does dissolve if I heat the solution to >55ºC.
The drug is dissolved in S-basal, then used at a final concentration of 100µM drug and 0.5% DMSO.

Any tips greatly appreciated,

Thanks!

2

Hi,

Your description is a little unclear, do you:

  1. first dissolve your drug in DMSO and then use dilutions of this stock in your experiments (so that DMSO final conc. is 5%) and it is at this point that the drug precipitates?

  2. first try to dissolve your drug in S-basal and just shove in a little DMSO for luck?

Letting us know what the ‘drug’ is would also be very useful.

Steve

3

Sometimes people include a small % of mild detergent like Tween-20 to keep things wet and happy as well. The worms don’t seem to mind.

4

Thank you for the suggestion kevinem . I have tried triton-X which I think is similar, but I’ll give tween-20 a try too.
I am trying to inhibit the proteasome using MG132. Final DMSO concentration is 0.5% DMSO/S-basal. 5% DMSO noticeably affects behavior so I have avoided going this high. I do of course dissolve the drug in DMSO as a stock solution, then use this in S-basal. Whether I add DMSO+drug to S-basal or vice versa, at room temp the drug precipitates.

5

Yes, 5% DMSO is too high, but 1-2% should be okay. I would steer away from Triton X-100 or NP-40 (or it’s alternative), those detergents are much ‘stronger’ than Tween-20.

6

and you have tried the steps suggested by Sigma and other Distributors? e.g. higher conc in DMSO so that there is a low final conc. in medium (0.2%) and warming BOTH medium and stock to 37-40 degrees before mixing them together?

If this is still problematic, you may have to play around with your incubation buffer composition.

7

I don’t have experience with MG132 but what about spreading it on the surface on NGM plates instead of mixing it into the medium?
This of course involves the problem of not knowing the exact concentration the worms are exposed to (how much of the drug stays concentrated
on the surface and how much diffuses into the NGM?), but the same is true if an unknown fraction of your drug precipitates in the medium.
Depending on your drug, ethanol rather than DMSO might be useful for preparing the dilutions to spread on the plates.
Good luck!
Peter

8

A number of labs have published studies using MG132 in worms (see https://scholar.google.com/scholar?hl=en&q=elegans+mg132&btnG=&as_sdt=1%2C21&as_sdtp=). Have you considered contacting them directly for guidance?