it should be no problem, in general, you should at least dilute more than 10 times. For example, in 2ml media you can use till 200ul, Good luck!

It should be as equivalent concentration as used in your extract.

You have to use the same amount of Methanol as you're using with the dissolved plant extract. If your plant extract is something like 50 ul, you have to use 50 ul Methanol as control - if you're using 100 ul, you have to use 100 ul Methanol to rule out the effect of Methanol. And basically you should not ask for the minimum Methanol concentration but for the maximum Methanol concentration your tissue culture can tolerate - and normally this will come in the range 0.1-1%, if you're going much higher, cells will start dying because of Methanol toxicity.

thank you very much for all. and thanks for the correction Daniela i want to ask the same that you explain. can i get some reference for maximum methanol concentration that tissue culture can tolerate?

I don't have any specific reference, 0.1-1% is based on experience and might differ from cell type to cell type. Personally, I would use a concentration of your plant extract in a way that you can go for a 1:1,000 dilution of the extract / Methanol as 1% might be already too high. And to really learn about your specific cell type - go for a titration using different dilutions and a simple trypan blue staining and cell counting after something like 24 hours. Might be a 2 days work but I think you can learn about your cells right a lot.

I don't think that a 1:20 dilution as you're using is really healthy for the cells. To prove this, one option is to have an extra cell or bottle without any treatment - this is my standard to rule out the effect of any soluble (like DMSO, PBS+BSA, Methanol etc.) when I am performing experiments.

Thanks for your advice. Do you have any protocol how to do toxicity test of plant extract on cell culture. Should i need to dilute my extract in water or something? 1st dilution in methanol and then in water?

I would not use water, I would use either PBS or cell medium to make a dilution for your second dilution. Or dilute the plant extract further with Methanol to obtain a concentration that you can go for something like a 1:1000 dilution.

thanks Daniela for your valuable answers

I am also using 60% ethanol extracts in my experiment. Initially I dissolved 30 mg of the extracts into 300 microlitre of 60% ethanol to make the stock solution and then I would further dilute it to 1:1000 or more to make the desired concentration with DNAase/RNAase free water or PBS and so on when I would use in in vitro cell culture. After the dilution I would also sterilize it with 0.45 micron injection driven filter before mixing with media. Now I have stored this solution (ethanol dissolved stock solution) at cold tempereature until further use.

Dear Daniela ! Thank you so much for your clear, straight forward explanation of methanol usage. I am just about to star my experiment and I have tried diluting Beta sitosterol - Stock Solution -  in many different concentrations of DMSO and or DMSO / Ethanol. I will proceed with methanol (respecting the ranges you have mentioned) with an information that really made my day !  

All the best success for you and God bless you

Peace and Health

Gus Amorim

RMIT Uni Melbourne / UFES Brazil

 i am using methanol in pbs  as solvent for preparing bsa(bovine serum albumin) protein solution as the another drug am using is soluble in methanol. Does methanol brings the structural changes in bsa??

Hi Erum !

If I understood right you are testing 100uL of your crude plant extract dissolved in pure methanol for a 2ml well (6 well plate?!). This is equivalent to 5% final concentration of your solvent ( methanol) per well.

  I suggest you to dilute your plant extracts in a way that the final concentration of methanol reaches 0.25 - 0.1%. I believe it would be healthier for the tested cells and you would have a much clear view of your extract's effect.   

When you make your serial dilution of the crude plant extract, be aware of the final concentrations you wish to test.

About Bhawna's praxis of using methanol / PBS mixture as solvent for BSA, I believe it is advisable to follow the same logic, e,g, try to use very low conc of methanol because in general  alcohol interact with tertiary proteins bringing some structural changes as a result of the denaturation process (disruption of covalent , non covalent dipole- dipole and Van der Waals interactions). 

I hope it helps.

thanks Mr. Gustavo, but lower concentration of methanol will lead the drug stay  insoluble. what protocol should one follow to see interaction between any protein and any compound that insoluble in water. What are other suitable solvents for dissolving protein other than pbs that dont affect its strucutre. How to deal with solvent selection for such system?

Ms Bhawana, you are correct on ur part that drug may precipitate by dilution as cosolvency works in higher concentration. The drug have certain solubility in water or any media in microgram/ml level you have to check it doing solubility studies. 

Here in this part you can get help of methanol in solubilizing initial amount of minimum weigh-able drug. And doing serial dilution with water/buffer to achieve its standard solubility and then you can see the effect of the drug.

For achieving higher concentration of solubility you can go for DMSO whose little higher concentration is permissible. But anyway same issue will be there.

 Thanks  Mr. Oostingh and Mr. Paswan