Drug recrystallize when i added to DMEM - (Aug/24/2012 )

Hi All,
I would like to thank the moderator to merge our questions.
My drug is an androgen analog and the Media is DMEM. However, as I mentioned before, I cannot see any drug crystals when I make 10 nM solution using my DMSO/Chloroform stock (however if I go more than 400nM the drug starts to precipitate). I am just wondering if the drug is precipitating and I cannot see it.
Thanks

Dear Xabi,
thank you very much for your valuable suggestion, i tried that, my drug was dissolved in 3 % Tween 80.

now i have several doubts to ask,

1. Can i autoclave the tween 80?
2. if is it possible to autoclave, may i prepare 3 % tween with DMEM for dissolve the drug ?
3. If it is not possible to auticlave tween 80, can i filter sterlize the 3 % tween 80 ?

looking for your kind reply.

thank you,
micronagu

micronagu,

first a question, how did you do to disolve the Tween80? Preparing a solution and adding the piperine directly or adding it from the stock made in DMSO?
My idea is to add a desired amount of the piperine in DMSO stock to the medium (water for testing) with Tween. But be sure that the medium has already the surfactant or you may not be able to redisolve. Try to get the minimum amount possible of Tween as cells are commonly quite sensitive to surfactants.

Tween80 is autoclavable, though the appareance of the medium after autoclaving looks weird... I don't know how to explain it... looks to be something wrong with the medium because the Tween80 partially separates from solution at high temp.

I don't know if piperine is autoclavable. I used to add the PAH to the medium with Tween80 before autoclaving because they are very stable. If not, and being more secure add it after cooling the medium.

Ambinlab, probably you are over the max. solubility at 400nM but not at 10nM. But it would be also possible to have some precipitate without being easily seen. Did you check it under microscope? If you cannot see anything under the microscope it's OK.


I just read that sometimes, the formazan is disolved using an acidified solution of 10-20% SDS (surfactant!). But the CMC (critical micelle concentration) of SDS is much higher, 0.23% (w/v). Depending the final concentration of SDS by this way of adding the formazan, it could be a way to solve the problem but SDS is know to be usually more toxic than Tweens, saponins and others at the same concentration.
Additionally, it seems that Tween80 may suffer autooxidation, so I would also test the interaction with formazan in case they can react giving false positive.

Dear xabi,

thank you for your reply.

First of all, I made 200 mM stock piperine solution with DMSO. Then I made 2 mM piperine working stock with 3% Tween80. Then after made piperine working stock with 3 % Tween, i would like to filter sterilization instead of Autoclave. because some of my friends suggest me its better to filter the Tween instead of autoclaving.

then from filtered working stock, i would like to add the to the cells for MTT assay. the maximum final concentration of the tween is 0.1 % and maximum DMSO concentration is below 0.01 %..

this is the way i plan to do my assay.. in your view, is that any thing to change in plan ?

Looking for your kind reply.

micronagu