We are measuring total cholesterol levels secreted into the media by cultured astrocytes using the Abcam cholesterol assay kit (ab65390).
Because the number of cells differ between wells, and the hemocytometer is not very accurate, we are trying to normalize the total cholesterol values.
It was suggest that measuring total protein from the cells themselves, and normalizing: total cholesterol/total protein would be a possible idea.
The premise is that the more protein you have, the more cells there are.
Does this make sense?
Are there other suggestions?
Yes, what you suggest is feasible. An alternate approach would be to stain your cells (left in the wells after harvesting the sups) with a nuclear stain like Hoechst 33342 and then read the fluorescence using a plate reader. The signal would be proportional to the number of cells you have in the well. If you rinse the cells with PBS before adding the staining solution, you may get rid of any dead/floating cells that may confound your results. The technique should take less time (
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