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Serially diluting compounds while keeping DMSO concentration the same

DMSO serial dilutions stock compounds

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#1 cellculture_newbie

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Posted 09 December 2011 - 01:09 AM

If I have a compound made up in a stock solution of 50mM in 100% DMSO and I make working solutions of 100uM in media, than that would reduce my DMSO concentration to 0.2% right? (please feel free to correct me if it is not 'cause this stuff is COMPLETELY new to me)

Now if I wish to serially dilute this compound in two fold dilutions (let's say in a 96-well plate), then I would do so by adding 100uL of the working solution to 100uL of media. However, wouldn't this also reduce my DMSO concentration? Theoretically if I just wanted to reduce the concentration of my compounds but maintain the same final concentration of DMSO in the wells, how would I go about doing this?

I would appreciate any help on this! Thanks so much!

#2 Rute

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Posted 09 December 2011 - 07:18 AM

I don't have much time now but tomorrow i can give you the exact numbers.

But yes, you are diluting your sample. What you need to do ins calculate the DMSO concentration after diluting (use formula: Initial concentration*initial volume=final concentration*final volume) and add dmso so that it stays the same as the inicial concentration.

Sorry if it wasn't much of a help.

#3 casandra

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Posted 09 December 2011 - 07:50 AM

If I have a compound made up in a stock solution of 50mM in 100% DMSO and I make working solutions of 100uM in media, than that would reduce my DMSO concentration to 0.2% right? (please feel free to correct me if it is not 'cause this stuff is COMPLETELY new to me)

Now if I wish to serially dilute this compound in two fold dilutions (let's say in a 96-well plate), then I would do so by adding 100uL of the working solution to 100uL of media. However, wouldn't this also reduce my DMSO concentration? Theoretically if I just wanted to reduce the concentration of my compounds but maintain the same final concentration of DMSO in the wells, how would I go about doing this?

I would appreciate any help on this! Thanks so much!


But which are you testing…your compound or the DMSO?
We use DMSO (or other solvents) if the drugs or chemicals don’t dissolve in aqueous solns but because of its cytotoxic effects, we usually try to keep it at a final conc of 0.1 %. Some have used it at 1% so it depends, as long as one runs the appropriate controls and the effects observed in the cells are not due to the DMSO. The further diluting out of the DMSO is actually preferable (some cell lines may be more sensitive to it) and hopefully your compound doesn’t precipitate or come out of solution.
"Oh what a beauteousness!"
- hobglobin, personal comment about my beauteous photo......

#4 bob1

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Posted 09 December 2011 - 03:22 PM

While I definitely second Casandra's advice, if you want to keep the DMSO the same - make the dilutions up in DMSO!

Edited by bob1, 09 December 2011 - 03:23 PM.


#5 cellculture_newbie

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Posted 09 December 2011 - 07:08 PM

Rute Ferera, thanks so much, I would appreciate any help I could get, I understand the logic and some sources have also suggested using M1V1=M2V2 but I think it would be a lot clearer if you could show me how that keeps the conc of DMSO the same using numbers.

#6 cellculture_newbie

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Posted 09 December 2011 - 07:15 PM

Hi Cassandra and bob1 - thanks for your reply. I'm testing the effects of the compound on my cells but I'm trying to keep the final DMSO concentration the same (but low) in all the wells. Isn't it important to keep the DMSO concentration the same in all the wells, or can we just run controls of DMSO to ensure that it's not killing the cells?

I understand that I have to make the serial dilutions in DMSO to keep the final concentration the same but I'm just finding it a bit difficult to do that using numbers. I am a complete noob in every sense of the word esp when it comes to dilution calculations!

#7 bob1

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Posted 11 December 2011 - 12:05 PM

You should definitely run what is known as a "Vehicle control", which is used to control for the effect of the diluent (in your case DMSO) on your cells. The vehicle comes from the fact that the DMSO is the "vehicle" that transports the drug. Technically you should run one for each concentration of the vehicle, but in most cases you can simply use the highest concentration of the vehicle as the control, as lower concentrations should have less effect.

0.2% appears to be correct for your calculation of the DMSO concentration.

The C1V1=C2V2 thing works literally as it says - C1 and V1 are concentration and volume you have now, and C2 and V2 are concentration and volume you want. If you know any 3 of the variables you can re-arrange the equation to work out the 4th. Just remember to keep the units the same!

e.g. You have 50 mM compound (C1) and want 100 uM (0.1 mM, C2) in 200 ul medium (or whatever volume you like, V2)

So now the equation looks like this 50 (C1) x V1 = 0.1 (C2) x 200 (V2). Rearrange to make V1 the subject of the equation:
V1= (0.1x200)/50
V1=20/50
V1=0.4 ul So you would take 0.4 ul of your 50 mM stock and add it to 199.6 ul of your diluent to make a 100 uM solution.

A hint that you might like to use - if you want to keep the DMSO concentration the same in all wells, dilute the drug in DMSO for you dilution series, but make it say 2x or 5x or 10x what you would want, then add a corresponding amount to each well with the medium. For example, instead of 100 um and adding 200ul, you could make 200 um (2x) and add 1/2 the volume (100 ul) to each well, making up the remaining 50% with medium.

#8 cellculture_newbie

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Posted 12 December 2011 - 11:34 PM

bob1, thanks SO much that makes sense. i'll definitely try that.

Also, I was playing around with some calculations and just want to check something ;

If I took 100uL of 0.2% DMSO (made up in media) and added that to 100uL of 0.2% DMSO (also in media), that would give me 200uL of 0.2% DMSO in media? And if I transferred 100uL of that mixture to a further 100uL 0.2% DMSO made up in media, that should give me the same concentration of DMSO?

If I had my compound in the first 100uL media too, than that serial dilution should effectively dilute my compound in half with each dilution while keeping the concentration of DMSO the same in each well? If all the above is true, that it would mean that I've (finally) got it.

#9 bob1

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Posted 13 December 2011 - 11:32 AM

You are correct on all counts.

#10 cellculture_newbie

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Posted 13 December 2011 - 04:17 PM

bob1, YOU JUST MADE MY DAY! Posted Image Thank you!!!





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