I want to see the change in the cholesterol level in my cells after the treatment. I am using the SH-SY5Y cell line.
Hi Ibrar,
there is a dye that can selectively bind cholesterol in cell membranes. Its called Filipin and you can easily use it in FACS after fixing your cells. So you dont even have to lyse them. Of course this will give you a relative and not an absolute measure.
Hope this helps.
Hi, if you are more interested in absolute measures, you may employ the fluorimetric assay by Molecular Probes (Amplex Red Cholesterol Kit). This assay seems to be extremely sensitive and easy to handle. Should work with fluorimetric or alternatively a colorimetric readout.
Colorimetric methods based on absorption of a dye also work. They are easy to use and rather cheap. See attached paper.
Some other methods (GC-mass spec) and ways to avoid artifacts in the fluorometric assay are described in: Robinet et al., J Lipid Res. Nov 2010; 51(11): 3364–3369.
doi: 10.1194/jlr.D007336
I can confirm that Amplex Red as well as filipin work well and are reliable.
Cayman Chemicals do an excellent easy-to-use cholesterol fluoremetric assay kit.
item number 10007640
link is below
https://www.caymanchem.com/app/template/Product.vm/catalog/10007640
I used Filipin III on IIF and you need to be able to excite the probe at 340-380 nm plus filipin III bleach and oxydate very fast, in the end I was able to visualize it using a multiphoton microscpe to reduce bleaching. Usually the FACS do not have such a laser as the violet excite at 405 and unfortunately the Filipin is not excited at that wavelenght. For the colorimetric assay, you have to determine if you are interest in total cholesterol content or unesterifyed free cholesterol, there are kit for both.
For the users of the Amplex Red kit, did you lyse the pellets directly in the kits reaction buffer or did you do an extraction? Thanks!
Halley Oyer. Do you have a response?
"For the users of the Amplex Red kit, did you lyse the pellets directly in the kits reaction buffer or did you do an extraction? Thanks!"
I am also interested in learning how you lyse the cells for this kit.
Thanks
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