QuantiFluo™ Cholesterol Uptake Assay Kit

Protocol SDS

Application

• For quantitatve determination of cholesterol uptake in cells and screening of modulators on cholesterol uptake.

Key Features

• Convenient. Treat cells directly in 96-well fluorescent plate.
• Safe. Non-radioactive assay.
• High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day

Method

• FL485/535nm

Samples

• Adherent cells

Species

• All

Procedure

• Assay takes 24-72 hrs, hand-on time 1 hr.

Size

• 100 tests

Shelf Life

• 6 months

More Details

CHOLESTEROLis a sterol and lipid present in cell membranes, and is transported in the bloodstream of all animals. It is used to form cell membranes and hormones, and plays important roles in cell signaling processes. Cellular regulation of cholesterol levels is a complex system in which irregularities have been tied to obesity and heart disease. Increased cholesterol uptake has also been linked to highly proliferative cancer cells. Through monitoring cellular cholesterol uptake, one can explore these growing health problems and screen for possible drug treatments. BioAssay Systems cholesterol uptake assay kit is based on cellular uptake of a fluorescently tagged cholesterol probe. The fluorescence intensity measured at λex/em = 485/535 nm is proportional to the amount of cholesterol uptaken by the cells.

The Positive Control isn’t working?

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.

I’m not getting the results I’d expect?

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.

There is no difference in RFU values between wells or RFU values are irregular?

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.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.