As far as I understand your technical procedure, you just add 50ul of your stock solution of a drug (dissolved in 100% EtOH) to your cell culture dish (with cells, right?), and then you wait for achieving the "final" concentration. Please correct me, if I got that totally wrong. I think such a procedure would allow some of your cells get in contact with both, high concentrations of EtOH, as well as high concentrations of your compound of interest. For many reasons (including desensitization, i.e. opposite effects of your working drug) it may be better to first dilute your 50ul in 5ml of media and then change the media, if this may produce too much stress (and changes) to your cells, you may dilute your compound (and the EtOH) in some of the media first and then add this to your cell culture dish; for example, you could use a few Milliliter (2.5 of 5ml) of your cell culture medium from the dish, mix the drug into it and then add this back. this may avoid any to high concentrations to your cells (not sure if this is critical at all, just in case). Of course, there could be negative effects of my advice: the drug could bind and adhere to the plastic material...

If you treat control cells with 1% ethanol, you will have your answer.  Alternatively, if your drug is soluble, create a 10-100mM stock and ethanol is irrelevant. 

Also, 5ml must be pre-equilibrated with CO2 because the volume is high.

I have never worked on this cell line. I've only worked on Vero cells (which are very resistant) but I think it is the same.

You should firstly evaluate the cytotoxicity of ethanol on your cells (on a wide range of concentrations, i.e. from 0 to 20%). On Vero cells the CC50 of ethanol is approximatelly 10%. Secondly, you should do a blank: cells+media+ethanol (1%). Use ethanol as a drug and compare.

Many articles deal with the toxicity of ethanol, but I think this effect depends on the cell line.

Have you done any control assays? you can have 2 controls for this experiment: media only and media plus 50ul of ethanol

A direct effect of 10 promille EtOH on isolated cultivated cells is difficult to predict (while it kills a human). I once tested up to 10% EtOH on the growth of 3T3 mouse fibroblasts by BrdU incorporation into newly synthesized DNA and found no difference over several days. However, I would expect that e.g. gene expression and certain metabolic and signalling pathways will become affected.

You could prepare a stock with a higher concentration and prepare an intermediate dilution in medium to further minimize the final EtOH concentration. Then, include 2 control experiments: One without drug (solvent only) and another one without drug and solvent, to validate your data (as you want to see drug effects and not a possible ethanol intoxication of your cells).

Upto 5% ethanol final concentration is acceptable to studies and tolerable to cells. Easy to

perform a trypan blue

Hi, Salman,

you can try first to prepare higher concentration of the stock solution: 10 mM, for example.

second, you should always have a control, ea. in the case if you are not sure about effect of the solvent, you haver to sak similar amoiunt of the solvent to the control cell. Better, of course, have one more control: without solvent. 

Plesae, also consider, that solvent (etahnol in your case) may have not only cytotoxoic effect (cell damage/death), but also effect of cell line development (without visobly effect on viabilty). In this case you will study not effect of the drug, but how this drug interact with effcet of the ethanol.

So, finally, plesae, test very carefully effect of the ethanol (1% as you say) to your cell line. Not only in the term of cell death, but in the term of cell growth/development.

Do you know any other organic solvent you can use? May be DMSO? In the case of DMSO, the concentration less than 0.1%  (1µl/ml) in practice does not have any effect on the cell. If you can prepare 100 mM stock of your drug, it definely solve the problem. 

My two cents..pure ethanol is hygroscopic and thus for all intents and purposes I calculate the stock to be at 95% rather than 100%. Unless I have just opened the bottle from a supplier that uses inert gas to top up the head space.

As far as I understand your technical procedure, you just add 50ul of your stock solution of a drug (dissolved in 100% EtOH) to your cell culture dish (with cells, right?), and then you wait for achieving the "final" concentration. Please correct me, if I got that totally wrong. I think such a procedure would allow some of your cells get in contact with both, high concentrations of EtOH, as well as high concentrations of your compound of interest. For many reasons (including desensitization, i.e. opposite effects of your working drug) it may be better to first dilute your 50ul in 5ml of media and then change the media, if this may produce too much stress (and changes) to your cells, you may dilute your compound (and the EtOH) in some of the media first and then add this to your cell culture dish; for example, you could use a few Milliliter (2.5 of 5ml) of your cell culture medium from the dish, mix the drug into it and then add this back. this may avoid any to high concentrations to your cells (not sure if this is critical at all, just in case). Of course, there could be negative effects of my advice: the drug could bind and adhere to the plastic material...

Of course has it an effect. What you also should have in mind is that it will have an effect on the Osmolarity of the medium. In our cases it decreased it even if we only cleaned the incubator with 70% EtOH if you add 1% in your cell-solution the effect could be stronger.

Then what should be the conc. of ethanol, that is not affecting cell line? As DMSO is safe at 0.05% conc.

The answer is: nobody knows, since this may differ from cell line to cell line, perhaps some neuronal cells or embryonic cells may be more sensitive and others not sensitive at all. As suggested in the first answers you may just do your experiment and include as control the identical procedure for adding your compound of interest within the 50ul of 100% EtOH and  as "negative" control the same amount of EtOH without the compound (best control would be some "inactivated", or any other irrelevant compound, but for this experiment you want to see the effect of your compound and exclude any side effects of the EtOH. Since I do not know anything about your experiments, you may think also of different concentrations (dilutions, dose curve) of your compound of interest.

I agree with Robert Eibl, it varies from cell to cell.

But, I have tried alcohol as solvent for my molecule, and treated cells; RAW, THP1 as long as ~48 hrs.

I first checked the maximum percentage of alcohol I could use with those cells, and with my observations cells were fine ~2% final alcohol. it may also depends on amount of media used with same number of cells. 

However, It may vary with your cells but I guess 1% could be in safer limits. You should first check the effect of alcohol on viability of cells before getting into experiment.

People regularly use DMSO, DMF and BME with their cells, and it seems a certain percentage of organic solvent is safe in terms of treatment.

Best

I support Saurabh's suggestion of starting with a kill curve for alcohol on your specific cells, but I would add a cautionary note. Even if 1%(v/v) ethanol is not toxic to your cells, you cannot assume that it is not bringing about an interaction with the drug you are testing - either enhancing or surpressing its effect. 0.1% (v/v) is a high enough concentration to see many cellular effects of alcohol, and 0.08% (w/v) in blood is considered legally drunk in many countries.

There is an additional complication which is that 1% by volume is a significant amount to add to your culture medium. Mixing is not instantaneous, and you may expose your cells to a much higher local concentration of vehicle in a manner that can vary substantially between dishes (or wells). I am never happy using anything less than 1000-fold dilution with organic solvents to living cells.

Are you sure that you cannot use an aqueous vehicle? Compounds that are insoluble at neutral pH can be soluble in dilute acid or base, in amounts that are easily buffered by standard culture media. Additionally, carriers (such as delipidated albumin or cyclodextrins) can be used to deliver hydrophobic drugs in aqueous solution.

You are right to be concerned about your vehicle. It is an often overlooked and frequently confounding aspect of drug effects.

I agree with Saurabh Srivastava .One thing more you have to check the effect of the cell line when treated with ethanol before starting an experiment on the same cell line .

Thank you all for your Nice suggestions. Actually in a research article (http://www.ncbi.nlm.nih.gov/pubmed/21738712), it has been mentioned that final conc. of ethanol could be 2%, anyhow they have used neuroblastoma cells, which are different form my cells line. I am looking for more suggestions from you people and i am really grateful to you.

Best Regards! 

Biswapriya Biswavas Misra, Thank you for your comment, do you have some reference,please, about 5% final concentration of ethanol? If yes, it will make me assure. Actually my drug has a greater solubility in ethanol as compared to DMSO, secondly, ethanol does not freeze, when it is stored at -20°C, while DMSO has to be melt before every use; causing irritation. 

The best thing to do is to include an ethanol control and an untreated control. You will be able to see how ethanol affects the parameters you are interested in. I sometimes treat cells with drugs dissolved in ethanol, usually using a 1:1000 dilution of the ethanol stock, which does not seem to affect the parameters we are looking at, but even so, we always include an ethanol control.

Given that most places in the US have a blood alcohol limit of 0.08%, I would guess your cells are a little drunk with a 1% ethanol milieu.

Good luck with your research!

You really should include an acceptable control. I remember some students in a neighboring lab who thought they had great results in their experiments on cell cultures, they could "reproduce" their finding, but it took years to find out that all effects where caused by "slightly" different handling of their controls: they left their control cells untouched in the incubator and washed and treated only their other cells with their compound of interest. The whole effect was related to the different, gentle shaking of the cells. They could have avoided wasting years of research with just the proper control, including the identical procedure and adding for example a negative control with washing those control cells with PBS, just shaking the cells the same way... You may have to compare your treated cells with untreated cells, it may be up to the reviewer to judge how good your control is.

make the final concentration less than 0.5%. 

In my experience with various cancer cell lines (but not yours), viability is not affected by 1% ethanol. But to be sure, you can test your cell line's viability with and without ethanol 1%. Or you could increase your stock solution to 10 mM, which will reduce the ethanol control to 0.1%.

A change in viability (trypan blue exclusion test) would show there is a big effect of the EtOH, but no change in viability does not at all exclude any other changes in pharmacological reactions to a compound of interest.

Another consideration. ..  Low vapor pressure solvents add Increased variation to assays because of evaporation of solvent each time the drug stock is opened.  Try to find a salt of the drug. 

and some biologically active substances do not like repeated freeze-thaw cycles. OK, your substance is in EtOH and not "frozen" under your conditions, but changes in temperature may not be tolerated by some substances. Lucky me, some of my plagiators did not know that and lost a year with old substances...

With mine and others experience, less than 0.5% of ethanol is kind of safe. However, it may vary from cell to cell and even this may be high for some cells.

I'm doing a similar thing where I am applying a drug to a cell line but it is dissolved in DMSO instead. In my 6 well plate I have a vehicle control well which has media with the same concentration of DMSO as the drug treatment well has. I count the cells for viability with Trypan Blue and because I'm doing cell cycle analysis for the drug's effect, I do the cell cycle analysis on the vehicle control as well to make sure the effect I'm seeing isn't from the DMSO.

Perhaps whatever analysis you are doing on the drug treated cells can be done with vehicle control treated cells as well? That way you know it's not an effect of the ethanol. Hope this helps!

I'd agree that a vehicle control is the sensible solution to this. Relatively small concentrations of solvents can impact on your measurements (for example, in my hands, 2% DMSO increased cell death by ~25% compared to 1% or no DMSO - although in other cell lines these effects may not be so great), and it's important to separate these artefacts out from the true data.

very small changes can result in failure of an experiment. I remember over 20 years ago when it was a big challenge to grow mouse embryonic stem (ES) cells, either on fibroblast feeder cells or with LIF (leukemia inhibitory factor) for K.O. (knock-out) experiments wiht HR (homologous recombination). It was necessary to grow those cells as good as possible to generate a whole new mouse and mouse strain from one cell, but often the normal looking cells did not succeed in germline transmission, although they remained "viable" as a cell line or contributed to chimeric mice. It always depends on what exactly one would like to see. For pharmacology tests it is reasonable to beleive that even minor changes with EtOH may change the response of some pathways. With the ES cell culture 2 decades ago it became crucial to use FBS with lowest LPS concentrations to avoid the lack of germline transmission.

I agree with the responses : you have to experiment with at least 3-4 Eth concentrations as this can vary in relation to kind of tissue, even if some indicated with 2%, no problem occurred.  As mentioned in the last commentary of Robert Eibi, slight variations in any compound or way to work could impact significantly cell growth.

I have always played safe and only used ETOH at 1 in 1000.  However,  I would suggest that you should try your cells with and without 1% ETOH or indeed a range of ETOH concentrations first before trying your drug and see what concentration is safe for your cells.  It really is the only way to be sure.  Good luck.

This concentration produces very small (5%) relaxation of the precontracted (phenylephrine 10-8M) arterial smooth muscle in vitro (rat aorta). exposed for about 10-20 minutes. What happens if it stayed longer and in the cell culture - I do not know. It definitely will have "some" effects. Even if ethanol would be

The best is to try. Make a pilot experiment comparing cell with ethanol vs cell w/o ethanol