I am currently working on a fluorescence based assay to determine the effect of certain compounds on cholesterol efflux in THP-1 derived macrophages. I use 3-dodecanoyl-NBD-Cholesterol for my experiments.
First, cells are labeled with NBD-Cholesterol for 24 h, then equilabrated with serum-free media and testcompounds for 18 h followed by incubation with Apo A1 serving as cholesterol acceptor for 2 h. Then, I aspirate the supernatant and transfer it to a fresh 96-well-black plate. The remaining cells are then washed 3 times and lysed by addition of 0,1% Triton X for 20 minutes. Lysates are also transferred to a new plate.
When I start my measurements with the plate reader (PerkinElmer) the obtained results are not usable in terms of randomly low fluorescence intensities. Does anyone have experience with those fluorescence based assays and could help me out?
Thanks,
Jonas
By coupling NBD to cholesterol you will change the properties of cholesterol. Be careful with conclusions that are only based on NBD-fluorescence results. See also: http://pubs.acs.org/doi/abs/10.1021/jp406135a
Thanks for your answer! There are assays already established using NBD coupled cholesterol instead of the tritium-labeled. Studies have shown that NBD- and tritium-labeled cholesterol behave similiarily regarding cholesterol efflux. Nonetheless I appreciate your advice!
However, in my hands it does not work. I label THP-1 monocytes for 24 h with 1 µg/ml NBD-Cholesterol (I already checked uptake under a fluorescent microscope with some cells). During starving, treatment and efflux stimulation NBD-cholesterol seems to vanish in large part, because there is a very low signal when measuring with a plate reader. I regulary check cells under a miscroscope after starving and they look happy.
NBD-Cholesterol and tritium-labeled cholesterol efflux protocols differ in certain aspects, but the former can rarely be found. So I would highly appreciate if someone would share his/her experience with me.
Fully agree with Frits, in factt C6NBD acylated phospholipids can transfer as monomers from donor vesicles to target membranes. This shows the altered amphipathic properties of conjugating the hydrophilic NBD moiety to lipids.
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