The question is basically about doing the vehicle control test for DMSO in mammalian cell culture (CHO cells). A working strength of 0.1 to 0.5 % DMSO is generally advised to be safer for most cells while doing a plate based in vitro experiment.
My agonist stock is 10-2M, made in pure DMSO, and I am doing a serial dilution running from 10-5M to 10-10M in seum free media outside the wells (in appendorfs). Total volume I need for each dilution is 200uL/well in triplicate in serum free media. The assay is being done in a 96 well culture plate where my adherent cells are seeded in a total of 200uL/well of growth media. How to do vehicle control for DMSO for the highest conc (10-5M) in the particular triplicate? And do I also need to add DMSO to my control wells so as to see any effect of DMSO alone? How shall I determine the percentage of DMSO in the highest dilution well i.e. 10-5M ?
I will replace the existing 200uL/well of growth media with the same volume of drug media in a dose response cAMP determination assay but I need to know my cells are not exposed to toxic concentrations of DMSO and they fall well within the acceptable range. Any help would be appreciated!
There is more to consider than just the maths here. I would studiously avoid any situation in which you have around 10% DMSO as an intermediate dilution. It's all too easy to exceed aqueous solubility via this route and precipitate compound. If you start with 10mM DMSO stock then I would make the first into aqueous 1/100 at least. I would combine this idea with the comment above about keeping the same final conc of DMSO in all wells, (a VERY good idea). Therefore Step 1 would be to make an initial series of dilutions in neat DMSO. If you do this in micronic tubes / racks then this and all subsequent steps can be performed with a multichannel pipette. Step 2: transfer 10ul of each neat DMSO dilution to empty 1ml micronics. Step 3 Add 990ul of your culture medium to all tubes. This should ensure fast even dilution which avoids compounds exceeding aqueous solubility. Top compound conc is now 100uM. Now add 20ul to 180ul of cells. Top conc of compound is now 10uM and final conc of DMSO is 0.1%. This should be fine for most cells, especially CHO.K1 which are very robust, even if exposure is for 3-4 days. I would nevertheless include a zero DMSO control just for good practice, and to eliminate potential synergy with any other non-specific parameters.
To avoid all potential complications in interpretation of your observations involving DMSO, include control for DMSO alone at each dilution of your agonist. It will save you lot of trouble.
There is more to consider than just the maths here. I would studiously avoid any situation in which you have around 10% DMSO as an intermediate dilution. It's all too easy to exceed aqueous solubility via this route and precipitate compound. If you start with 10mM DMSO stock then I would make the first into aqueous 1/100 at least. I would combine this idea with the comment above about keeping the same final conc of DMSO in all wells, (a VERY good idea). Therefore Step 1 would be to make an initial series of dilutions in neat DMSO. If you do this in micronic tubes / racks then this and all subsequent steps can be performed with a multichannel pipette. Step 2: transfer 10ul of each neat DMSO dilution to empty 1ml micronics. Step 3 Add 990ul of your culture medium to all tubes. This should ensure fast even dilution which avoids compounds exceeding aqueous solubility. Top compound conc is now 100uM. Now add 20ul to 180ul of cells. Top conc of compound is now 10uM and final conc of DMSO is 0.1%. This should be fine for most cells, especially CHO.K1 which are very robust, even if exposure is for 3-4 days. I would nevertheless include a zero DMSO control just for good practice, and to eliminate potential synergy with any other non-specific parameters.
- Badges
- Science topic