I'm using methyl-β-cyclodextrin (MBCD) to deplete cholesterol from mammalian cells. I'm following the Mahammad and Parmyrd protocol, in which cells are treated with different MBCD concentrations, ranging from 0.5 mM to 2.5 mM to evaluate the level of cholesterol depletion. However, in the protocol description, they use 20 million cells in one ml, and add one ml of the desired concentration (0.5 mM to 2.5 mM of MBCD). Although the protocol advises not to deplete cholesterol in adherent culture, sometimes is needed depending on the experiment, as I have seen many authors do, but, the reported concentration in those papers is usually 5mM in adherent cultures as if they put a 5mM MBCD solution in medium in the plate or well regardless the cell number. Should I normalize to the cell number in my well or plate? or just pour a 5mM solution on the plate without considering the number of cells?
Thanks in advance
Dear Carolina, thank you for posting this interesting technical question on RG. Unfortunately I cannot provide you with a qualified expert answers because we are inorganic chemists. However, I can suggest to you the following relevant research article which might help you in your analysis:
Cholesterol Depletion Using Methyl-β-cyclodextrin
Article Cholesterol Depletion Using Methyl-β-cyclodextrin
This paper is freely available as public full text on RG. It has a detailed experimental section. Also please have a look at the following potentially useful article:
Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies
Article Use of cyclodextrins to manipulate plasma membrane cholester...
This paper has not yet been posted as public full text on RG, but both authors have RG profiles. Thus you can easily request the full text directly from one of the authors via RG.
Good luck with your research and best wishes, Frank Edelmann
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