I use plant extracts of varying concentrations from 1 ng to 10 ug for estimating lipids in 9 day old 3T3-L1 adipocytes by Oil Red O staining. The cells are treated with the extracts throughout the period of differentiation. I tried to analyse the intracellular lipid formation in adipocytes by using ORO. The quantitative part of the assay was good but the qualitative part of the assay was not satisfied, i.e., the cells are less intensely stained. I get the same issue whenever I try. Suggestions are welcomed and thank you in advance
Well I have never tested whole plant extract if you are asking me that. But I did test various lipophilic/hidophilic compounds on primary cell cultures and I did wash them from compounds successfully. In her question I saw the fact that the compounds in medium interact with dye and the replacement of medium is always needed although many researcher just add the dye in culture plate with compounds dissolved in medium. THAT is so wrong on many ways! Because you can never know what is the effect of the compound on dye solution.
The interactions with cell structures that you are mentioning is/are the mechanism of action of unknown tested compound. The inhibition of cell transport and/or some metabolic pathway is just the mechanism of action of tested compound and in my opinion it should be mentioned in publication. The results that she obtained are interesting and can be discussed in paper.
Yes, plant extracts in the incubation medium can interact with the staining. Either when they interact directly or when secondary plant metabolites had interacted with the cell surface, they can inhibit uptake of the staining dye.
Thank you very much sir. As secondary metabolites are concerned, I work on tannins of concentration 1ng to 10ug. Will it be rectified by decreasing the concentration?
Dear Snehaa, thank you. Yes, tannins are well-known for quite complex interactions. However, your concnetrations are already quite low, so I am not absolutely sure what to do...
Just prior to staining remove the tested compound from well and wash it! It should be OK after.
thank you very much Dr. Sasch Rohn
I will try it out sir@Nikola and thank you
Dear Nikola, are you experienced with that ? How would you wash it ? From our experiments, it seems to be quite diffficult to get rid off these compounds when once bound to proteins, cell surfaces etc...
Well I have never tested whole plant extract if you are asking me that. But I did test various lipophilic/hidophilic compounds on primary cell cultures and I did wash them from compounds successfully. In her question I saw the fact that the compounds in medium interact with dye and the replacement of medium is always needed although many researcher just add the dye in culture plate with compounds dissolved in medium. THAT is so wrong on many ways! Because you can never know what is the effect of the compound on dye solution.
The interactions with cell structures that you are mentioning is/are the mechanism of action of unknown tested compound. The inhibition of cell transport and/or some metabolic pathway is just the mechanism of action of tested compound and in my opinion it should be mentioned in publication. The results that she obtained are interesting and can be discussed in paper.
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