You may need to titrate the Positive Control to determine the optimal concentration for the cell line you are using. Titrate down from the recommended Positive Control concentration of 2.5 µM we suggest in the protocol.
The assay is a cell based assay which intrinsically leaves lofty room for variation in experiments. To account for variation amongst cells, we recommend running each variable in at least duplicate, however, preferably triplicate or greater.
Using high concentration (greater than 1%) serum media for the assay. Serum binds the Fluorescent Tracer making it inaccessible to the cells. Use serum free media or low percentage (less than 1%) serum media.
Not allowing the cells enough time to uptake the cholesterol. The cells need time to uptake the Fluorescent Tracer that is accessible to them.
Wells were not washed. If the wells are not washed after removing the media, some of the Fluorescent Tracer containing media may be left in the well. This can give positive bias and irregular results.
Cells were not aspirated carefully. When removing the media or wash buffer from the wells, be sure to pipette gently to avoid pipetting up cells with the media / wash. The Fluorescent Tracer uptaken by the cells cannot be quantified if the cells are not there.
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If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.
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