13 replies to this topic

[Ambinlab]

Dear All,

I just received a compound. In its brochure, it is mentioned that it is soluble in Chloroform. I made 1mM solution in Chloroform. If I add this to water/Media, chloroform won't dissolve (Stupid me!)

The trick that I did was to dissolve it in DMSO (100uM solution) and use that as stock solution and make 100nM working solution in cell culture media. I can't see anything precipitates and it seems to be quite stable.

I am wondering if my way of doing this will be OK. I thank you if you could comment on this.

Thanks,

[El Crazy Xabi]

I used to work with PAHs (phenanthrene, pyrene,...) and we disolved the compound in acetone. However if you added to a medium without a surfactant (I used Tween80), it appeared as cloudy due to thin precipitation but concentration was 100 uM in the media, 1000 times higher than yours. It also depends on your compound, medium,...

If you give details of compound, culture media,... would be easier to give a proper response

[micronagu]

hello every one,

i am working on organic drug which could be dissolve in DMSO. After i added the drug which is dissolved in DMSO stock, to the cells in DMEM for MTT assay, i have observed that the drug was recrystallized.

i have tried strong vortexing at 37 degree C, but it didnt dissolved completely.

I request you to please help me to figured it out..

Looking for your kind reply.

Thank you,

micronagu.

[ascacioc]

Guys, I merged your two questions because they are very similar (and came one after the other, please check the forum before posting).

Andreea

[micronagu]

for cell culture assays, the DMSO concentration should be same or below 0.01 %, hence i dissolve the drug in DMSO in higher molar concentration like 40 mM, and added to the DMEM medium that DMSO concentration should be prescribed. but even though the drug concentration was very low, it recrystallize, and i can see the crystals under microscope, when i was performing MTT assay.

I wish you to help in this issue and kindly help me suggest me how to dissolve the drug completely in the DMEM.

my drug was Piperine and i used this in microbiological and cell culture assays..

thank you,

micronagu.

Edited by micronagu, 28 August 2012 - 01:24 AM.

[DRT]

sometimes disolving in FBS or BSA will help

[El Crazy Xabi]

DMSO is ok to make stocks but if you have to add it to a aqueous medium it will happen the same as with the PAHs. To keep hydrophobic compounds/compounds with very low water solubility away to precipitate you will need something else: a surfactant, or any other substance that can increase the solubility.

A cyclodextrin may be one suitable surfactant for this, specially the cell cultures, as some are already in use in drug delivery. As DRT said, some sera can also help to keep it soluble.

[micronagu]

thank you Xabi, and DRT.

so, i would like to dissolve with surfactant such tween 20... so please tell me the concentration of DMSO and TWEEN ... it would be very useful to me..

looking for your kind reply,

thank you,

micronagu

[El Crazy Xabi]

If you use Tween20, you don't need to add DMSO by itself, it's added because you dissolve your compound on it. But you may want to add some extra DMSO to compensate the controls, so the DMSO in all cultures is exactly the same.

I used Tween80 at 1% but my compounds final concentration were 100uM and Tween20 is different. You may need test it and remember to add the same concentration to all media to control the possible toxic effects by the surfactant

[micronagu]

Thank you dear Xabi..

so, as you suggested me that,  I will going to use 1 % Tween 80..

From stock I will dilute my drug to 1 % tween 80 for preparing working stock.. from that I will add the drug dissolved in Tween to the cells. for control, same concentration of DMSO and Tween should be added. can you please tell the maximum percentage of Tween 80 could be not toxic to the cells.. ?

thank you,

micronagu

Edited by micronagu, 29 August 2012 - 10:13 PM.

[Ambinlab]

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

[micronagu]

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

[El Crazy Xabi]

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.

[micronagu]

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