I think you made the stock solution too low since the the solvent, DMSO, concentration becomes 0.5%. I suggest that you start with new vial of the drug or newly prepared chemical. Make sure that the stock solution concentration in DMSO should be well above 2,000X to reduce final DMSO concentration in the culture medium. For example, 2mM stock solution, aliquoted to 5 ul in small eppen-tubes, stored at -70C until use. When you use, add medium into the eppen-tube directly (495 ul medium make first dilution of 100X).

Spin it down 12,000 rpm for 5min after mixing well, you would see cloudy precipitation. In water condition, that is the maximum solubility of your stock solution. Take a necessary amount of supernatant into fresh medium for second dilution of 20X. Spin it down as you did in first dilution. Take and save the spernatant for the treatment group culture. In the second dilution, the chemical would salt-out making precipitation again.This is a proper way of water insoluble drug treatment. If you add your DMSO-drug directly to medium or any water for culturing zebrafish embryo, larva, or fry, the salting-out particles will damage the fry on any contacting outer and inner surfaces of the baby fish. Smaller particles such as nano or micro-sized will be endocytosed by epithelial cells of the skin, gut and respiratory organ that are opened to environment. The final concentration of DMSO would be 0.1% which in this case.

We make our stock solution of drugs 20,000X stock solution. In those cases, the final DMSO conc. is merely 0.01%. This lower DMSO allowed us to do longer exposure experiment, and more opportunity to find true drug effect without synergistic or hindering DMSO effects even if we have DMSO control group.

So, try higher stock as much as you can. Good luck to your trial!!!

Dear Sang Ho Lee, thank you so much for providing this valuable insight into the matter! I'll try as you say. Best!!!

Hi Agnieszka

I used to use the system water in 6hpf, 24hpf, 48hpf and 72hpf. I used to keep those larvae in system water for 7 days. It used to work well. You don't find any morphological changes due to lack of E3 medium.

It all depends on how many days your experiment plan is about.

With regard to solution preparation, I used to face problem of solubility when I directly take the drug and dissolve in 0.5% DMSO. So we used to take the drug into the 100% DMSO and then do serial dilutions. And we used to keep the final DMSO concentration to 0.1%. It used to work.

Dear Venkata, it's good to know that you didn't observe any morphological changes. I keep my 5 dph larvae in solutions only for a total of 2 h.

I prepared my stocks in 100% DMSO, then just did one dilution to the final concentrations and adjusted DMSO when needed to get 0.5% DMSO in each drug concentration. When I tried to get higher concentrations, solutions precipitated, probably because E3 components affected the degree of dissociation. This way I managed to get e.g. 20uM in 0.5% DMSO, but failed in case of 50uM. So, I'll do higher concentration of stock and serial dilutions, as you suggested. Thank you so much for your comment! Best!!