Please suggest me whether minimal amount of chloroform (0.1%) is suitable to use in cell culture experiments. As my organic compound shows degradation behavior in DMSO solvents, I prefer using an organic solvents to carry out my biological experiments. But there is another problem where organic solvents can't be miscible with cell culture media. So please suggest me any suitable organic solvents for doing cell culture experiments.
Thank you.
I am sorry because I misread your post, Amira. Who told you that 10% is the maximum?
You should always try to use as little amounts of solvent as possible. This is accepted to be true for all possible substances as far as I know (DMSO, DMF, EtOH...). In our lab we have the unwritten law to not exceed 0.1% wherever possible.
I know that you have higher amounts of DMSO in your solution if you freeze the cells and keep them in liquid nitrogen. But in this environment the cells aren't growing and they cannot metabolize the substance so you don't risk mutations. After thawing you have to remove the protecting agent as soon as possible.
Chloroform is not ideal. As you say, it is not miscible in water. I suggest trying ethanol instead. It is toxic at higher concentrations, but I have seen a number of studies using 0.1% ethanol without obvious effect. However , I would definitely trial it in your assay first to make sure it is ok.
Ethanol or isopropanol are usually fine, if it is less than 1% total volume.
Regardless what you choose, be sure to do some vehicle controls, to check the effect of the solvent alone in your cell and assay system.
Regarding DMSO, are you using fresh and clean DMSO? It's quite hygroscopic and most of my problems in the past with DMSO as a vehicle have been resolved by getting a fresh bottle.
If I have a compound which isn't stable in DMSO, I usually make up a fresh stock for each use. If you can't do that, then ethanol or isopropanol should be fine, as others have said. You might also get away with acetonitrile. Just be sure to use the appropriate vehicle control whichever way you go, and all should be OK.
Thank you Ian, Kamil, Peter and Mark for your valuable suggestions.
Peter: I used fresh DMSO even then I am facing a problem. I feel that is because of my compound stability.
Could you please explain me in detail regarding term "Vehicle control"?
A vehicle control is an experiment performed ideally in parallel with your drug/compound study using just the solvent, no compound. So you're controlling for the effects of the vehicle (solvent) on the cells or in the assay. Basically, it's a sham study. If your compound is quite precious, you may want to screen some vehicles (solvents) first, to see which your cells and assay tolerate best, then proceed with that as the vehicle for your compound.
Vehicle controls are particularly important in any cytotoxicity study, as the vehicle may cause some cytotoxicity itself, which you might blame on the compound you are trying to deliver, for example. Likewise, in studies involving membrane mechanics and channels these controls are important, as many solvents that can fluidize or otherwise affect the lipid bilayer, thus having effects of their own, amplifying or potentially masking the effects of the compound to be tested.
Thank you peter.
Basically my compound is highly soluble in Chloroform and Dichloromethane. In such cases, I make very high stock concentration with these solvents and again dilute with methanol. Finally I treat the cells which will subsequently have less percentage (0.1 to 1%) of these solvents in cell culture media. Is that okay?
sterile DMSO (stored in dark conical since it is light sensitive) of concentration of less than 10% is not toxic for the cell culturing techniques .
Even if 10% DMSO is not toxic for your cells it will still work as a strong mutagen and it decreases the activity of your cellular oxidational repair machinery by blocking MsrA enzymes. According to the literature (Kwak, BMB reports 2009) 1% DMSO is already enough to decrease the enzyme activity by >90%.
I am sorry because I misread your post, Amira. Who told you that 10% is the maximum?
You should always try to use as little amounts of solvent as possible. This is accepted to be true for all possible substances as far as I know (DMSO, DMF, EtOH...). In our lab we have the unwritten law to not exceed 0.1% wherever possible.
I know that you have higher amounts of DMSO in your solution if you freeze the cells and keep them in liquid nitrogen. But in this environment the cells aren't growing and they cannot metabolize the substance so you don't risk mutations. After thawing you have to remove the protecting agent as soon as possible.
We routinely use non-denatured ethanol with cell lines and freshly explanted hematopoietic cells. Some hematopoietic cells are surprisingly tolerant of ethanol. They can be cultured in 2% ethanol for several days. Others show signs of toxicity at ethanol concentrations as low as 0.2%.
It is difficult to predict what DMSO will do for you. In our hands, long-term exposure to 1% DMSO completely suppresses the growth of granulocyte/macrophage progenitors. Early erythroid progenitors don't fare much better. Late erythroid progenitors, however, love it; DMSO enhances colony formation more than 2-fold. In other words, DMSO can be more than a solvent. It may modulate the activity of physiological regulators or may itself have biological activity.
try to dissolve your product in ethanol and add in your culture medium at a concentration lower than 0.5%, if possible. Otherwise you may put your ethanol solution in the bottom of the culture flask and let it evaporate slowly . You will have no more organic solvent in your medium and the very low final amount of your product will be either dissolved in the aqueous medium or taken up by BSA
Because studying the effect of ethanol on the immune system has been a major topic of my research for many years, the idea of using 0.5% ethanol on cells is a little alarming. It probably has fewer confounding effects than a similar concentration of DMSO, but it still has huge effects on a wide range of signaling parameters (depending of course on stimulus and cell type). However, we have not seen major effects on anything immunological at levels of ethanol in the 0.05-0.1% range. Chloroform may not have major affects at their concentration on most cells, either, I just don't know. In any case, the best option is to compare untreated and solvent treated cells with regard to the parameter(s) you will be measuring in the experiments. If solvent doesn't affect the parameters of interest, but test chemical dissolved in solvent does, that should be a usable system-though it is still possible that the effect is mediated by a synergistic action of the solvent and test compound. One way to decrease the possibility that this is occurring is to confirm that a second solvent gives similar effects with the same test agent (it would be unlikely that two different solvents would synergies to enhance the effects of the same test compound in the same way).
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