I need to test various concentrations of a compound that is only soluble in 100%DMSO. My compound is in pure powder form (I have 1g available, and I will dissolve in 100% DMSO to prepare my stock). After that I want to dilute the stock to different concentrations but in the same time I need DMSO concentration to be fixed as 0.1% because I will try on cancer cell lines. How can I manage that.
Note:
the total volume of each diluted compound should be 1 ml
Hi Fatma Mohamed Fouad , in order to get fixed 0.1% DMSO in all concentration prepared, you need to add 1uL of your stock (compound +DMSO) to 999uL diluent at all time. In this case, doing serial dilution is no longer possible as the resulting subsequent dilution after your first initial concentration will have
Dilute your sample 1:1000 to get 0.1% DMSO. After that, just use a 0.1% DMSO solution as your diluent and dilute to any concentration that you like.
Hello @Fatma Mohamed Fouad,
Firstly, prepare a primary stock solution of the compound by dissolving it in 100% DMSO at a concentration that is 1000 times higher than the highest final concentration you want to test. Then, for each final sample volume of 1 ml, add 1 µl of this stock solution to 999µl of cell culture medium. This will give you the desired final compound concentration while keeping the DMSO concentration fixed at 0.1%.
For testing concentrations lower than the lowest achievable concentration using the primary stock, dilute the primary stock solution 10-fold or 100-fold in 100% DMSO to create secondary stock solutions. Then, add 1 µl of these diluted stocks to 999µl of medium to reach lower final compound concentrations. This helps in maintaining the desired final compound concentration while keeping the DMSO concentration fixed at 0.1%.
Therefore, in order to test various concentrations you need to prepare various primary stock solutions that are 1000x higher than the required final concentrations.
I hope I made myself clear.
Thank you.
That seems unnecessarily cumbersome. If you make a 0.1% DMSO solution to use as a diluent, you can dilute the 0.1% DMSO stock to any concentration that you desire. This eliminates the need for making multiple stock solutions.
Compound can be dissolved completely in 100% DMSO according to question. So using 0.1% DMSO as diluent might lead to incomplete dissolution. So your stock solutions will not be of accurate concentrations. Since cell culture studies can vary based on very minor differences in concentrations of compounds that are tested, using 0.1% DMSO as diluent would not be a good option.
And what diluent will be used to make 0.1% DMSO? If water then your media concentration might be less than 1x. If media itself is used, then to need more volumes of cell culture media with DMSO and media might interfere with dissolution.
I suggested dilution in 100% DMSO and then diluting to 0.1% DMSO as the stock solution. We do not disagree.
The diluent should be made from whatever solution is needed. For cell culture, use media as a diluent. The final concentration of the compound and DMSO will be the same as your suggestion, but a lot easier to prepare and a lot easier to adjust without having to make multiple secondary stocks.
Hi,
You may use the web tool to calculate
https://www.alfa-chemistry.com/solution-dilution-calculator.htm
https://www.alfa-chemistry.com/mass-molarity-calculator.htm
Alif Ismail, but if I did serial dilution in my solvent, then I would take, for example, 0.1 mL from this and add it to 100 mL of media. Then, my first concentration would change, right? @ Neeraj Kalyan Gandholi
Michael Marino do you mean that I should prepare 0.1 % DMSO from the stock from the beginning, then I can prepare my dilution from this new stock, as any DMSO concentration less than 0.1 will not affect my result?
Hi Fatma Mohamed Fouad
If you dilute using a different solution or solvent, then yes, your DMSO concentration will change.
In your case, you should perform serial dilutions of your original stock in 100% DMSO for each target concentration. This way, you’ll generate intermediate stocks with varying compound concentrations, but all in 100% DMSO. These will be your intermediate solutions for preparing your final working solutions while maintaining a fixed 0.1% DMSO.
For example, you can prepare a 100 mM stock by dissolving the appropriate amount of your compound in DMSO. Then, dilute 1:2 with DMSO to get 50 mM, then again for 25 mM, 12.5 mM, etc.
To prepare your working solutions, simply add 1 µL of each intermediate DMSO stock to 999 µL of your media. This will give you final concentrations of 100 µM, 50 µM, 25 µM, etc., all with a consistent 0.1% DMSO.
Hope this clears up your doubt!
Alif Ismail yes I understood you, but for serial dilution, I am still confused
Hi Fatma Mohamed Fouad,
No, I suggested making a diluent of 0.1% DMSO to make all further dilutions of your stock. So, for instance, if your stock is in 0.1% DMSO and you want to make a 1:100 or 1:1000 further dilution of your stock, but still maintain a final DMSO concentration of 0.1%, then you can dilute using a solution that also contains 0.1% DMSO. No matter how much you dilute, your final concentration of DMSO will still be 0.1%
Hi Fatma Mohamed Fouad . You have to be clear on why do you want the concentration of the DMSO to be exactly 0.1%. If you are worried about its toxicity, i think as long as your first initial dilution is 0.1%, subsequent dilution will brings the DMSO concentration down which will be no longer toxic for most cell lines. However, you should still include vehicle controls to account for any effects of the DMSO solvent.
Unless you have a specific reason to maintain a constant DMSO concentration across all treatment preparations (e.g., optimizing specific conditions), you don't need to worry about it too much.
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