The unidentified substance of my interest derived from a plant is only methanol soluble. how would I do MTT assay using this substance of my interest?
Dissolve it in methanol but do keep in mind that the final concentration of methanol should be less than or equal to 1% you can also use DMSO to dissolve your substance and here also the the concentration of DMSO should not exceed 1%
The substance gets dissolved from the lipid extract of the plant only when 90% methanol is used
So take that as your vehicle, but do remember it should not exceed 1% of the entire contents. You dissolve your substance like 1mg in 10ul of 90% methanol and then add 990ul of incomplete media of your respective cell line to make the drug with 1mg/ml concentration.
Good Luck!
The following answer is focused towards the cell culture assay not animal dosage. For animal feeding, obviously compounds should be dissolved in some organic solvent before administration.
I have a range of chemical compounds which are soluble, partially soluble and insoluble in water or media, however the partially soluble and insoluble compounds are readily dissolved in DMSO and some in methanol as well. Initially, I used minimum DMSO (say 4-5% of the final volume) for dissolving the compounds and used equal volume of DMSO as control. I was not satisfied with the results as all the readings were haphazard and not reproducible as well.
I used to observe that the DMSO dissolved compounds would rather get precipitated once I put it in the media for serial dilution.
The day I used all the compounds as such (without dissolving in any organic solvent) I got consistent results, that too dose dependent. Although, the higher concentration compounds would settle down in the tube but it seemed that they are somehow effective in that state also, there was no saturation observed.
The idea of using such method was based on some facts, such as:
1) Some compounds do not get dissolved immediately and take some time to get dissolved.
2) DMSO dissolved compounds tend to get precipitated in the media, that too unequally.
3) In nanoparticle studies, the entrapped compounds are never dissolved but present as discreet entities, still they show their effects, so why not the undissolved compounds be effective proportionately.
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