Fluorometric detection of free cholesterol in membranes of the living cells

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Fluorimetric detection of the free cholesterol on membranes of the live MDCK cells

Fluorimetric detection of free cholesterol on membranes of the live MDCK cells incubated with fluorescent cholesterol analogs for 10 and 60 minutes. The fluorescent cholesterol analogs used were; A) R-Chol, B) 22-NBD-cholesterol, C) 25-NBD-cholesterol. The figure D, E, and F are simply the high-magnification images of the A, B, and C respectively. Credit: Ogawa and Tanaka, 2016. 

Short description about cholesterol

Cholesterol is a sterol that constitutes the third most abundant lipids of the cell membranes. If you are wondering about the first and second most common membrane lipids then here is the answer. Phospholipids are the most abundant membrane lipids while the second most abundant membrane lipids are the glycolipids.


Cholesterol can be esterified (cholesteryl ester) and free cholesterol (non-esterified cholesterol). It is the free cholesterol that is mostly found in the cell membrane where it maintains the fluidity of the lipid bilayer. However, distribution of the cholesterol greatly varies among the various cell membranes.

Previously, various biochemical tests were used to measure the total cholesterol residues in the cell membranes and according to those biochemical tests, about 80-90% free cholesterols are found in the detergent-resistant fractions of the plasma membranes (DRMs). But, now a day, researchers have developed cholesterol analogs that can be used as fluorescent probes to localize and study the dynamics of the intracellular free cholesterols.

Fluorescent cholesterol analogs

The fluorescent probes are made up of cholesterols, some examples of which are as follows; dehydroergosterol, filipin, 22-NBD-cholesterol, 25-NBD-cholesterol, and BODIPY-cholesterol. Among these fluorescent probe cholesterol analogs, 22-NBD-cholesterol and 25-NBD-cholesterol are most commonly used. However, using these fluorescent probes, fluorescence microscopic examination shows that the exogenous NBD-cholesterols are hardly present in the cell membrane.

Therefore, to analyze the location and measure the dynamics of the free cholesterols on the plasma membrane, researchers developed a new fluorescent probe for the cholesterol on the cell membrane. They synthesized 3β-amino-5-cholestene, a fluorescent probe that they used to make new fluorescent probe-labeled cholesterol analog, 2-[3-dimethylamino-6-dimethylazaniumlidenexanthen-9-yl]-5-[2-[(5-cholestene-3β-yl) carbamoyl]ethylcarbamoyl]benzoate (R-Chol).


The researchers stained the live cells with this newly synthesized fluorescent cholesterol analog, R-Chol and then investigated the intracellular distribution of the cholesterol by measuring the fluorescent signals emitted by the R-Chol. The pattern of the signals emitted from the R-Chol was compared with that of the fluorescent signals from NBD-cholesterol-strained live cells. The comparison of the fluorescent signals was performed using the microscopy during which researchers found the presence of excessive free cholesterols in the plasma membrane.

MDCK cells were used for the study

For the study, researchers took Madin-Darby Canine Kidney Epithelial (MDCK) cells. They grew the cells in 96-well plates and washed twice with Dulbecco’s Modified Eagle’s Medium (DMEM) and then incubated the cells with fluorescent probe cholesterol analog (fluorescent probe dissolved in 10 µl ethanol supplemented with 50µl of 10 % Pluronic F127 and 9.94 ml of DMEM) for 10 minutes at 37°C.

After 10 minutes, cells were washed twice with DMEM and then again incubated in DMEM (supplemented with 10 % fetal bovine serum or FBS) for 6, 24 and 48 hours at 37 °C. After incubation, 10 µl of 10 water-soluble tetrazolium salt (WST-8) was added in each of the wells and incubated again for 1 hour at 37 °C under the 5% CO2 atmosphere.

The cellular dehydrogenase reduces the WST-8 to WST-8 formazan dye. The formation of WST-8 formazan dye can be monitored using the absorbance at 450/655 nanometer to measure the cell viability. The cell visualization was performed using the differential interference contrast and fluorescence images were taken using an IX70 microscope fitted with standard UPlanSApo and water immersion UPlanApo objectives.

The fluorescent signals emitted from the NBD and tetramethylrhodamine were using the U-MWIB3 and U-MWG2 cubes. The filters used were 460-495 nm and 510-550-nm band-pass excitation filters and 510-570-nm long-pass emission filters. They also performed pulse-chase imaging of the live MDCK cells incubated with the fluorescent cholesterol analogs in the same was as mentioned above.

Cell viability and localization of the fluorescent cholesterol analogs

Researchers performed cell viability test. During which cells were incubated with 0.5 to 50 µM of R-Chol for 10 minutes and the result was compared with that of the controls not incubated with R-Chol. However, no differences were observed in the morphological structure as compared to that of the control cells.

To determine the localization of the R-Chol, researchers stained the MDCK cells with the R-Chol or NBD-Cholesterol. Researchers observed the fluorescent signals and interpreted the data to find out the localization of the R-Chol. The NBD-cholesterols emitted the intense fluorescent signals around the perinuclear membranes (e.g. Golgi complex and endosomes) with very little in the plasma membrane.

However, in the MDCK cells supplemented with R-Chol, the fluorescent signals were emitted not only from the perinuclear membranes but also from the nuclear membrane and the entire plasma membranes. This indicates that the R-Chols are localized at the plasma membrane of all cells.

The presence of hydroxyl group is the most important part for the molecular recognition of cholesterol because it provides a site for hydrogen bonding with proximal molecules. This is the place where the fluorescent probe can be attached. For example, cholesteryl BODIPY FL C12, a fluorescent probe that is attached to the hydroxyl group of cholesterol promotes cholesterol localization on the cellular lipid bodies but not in the plasma membrane.

However, fluorescent cholesterol analog, R-Chol, localizes mostly in plasma membrane unlike the one mentioned a while ago. This is because R-Chol contains amide group that can form the hydrogen bond with proximal molecules in the same way as the free cholesterol forms.


The main problem with the BODIPY-cholesterol is that its distribution in the lipid bilayer is far slow as compared to that of the free cholesterol. However, this is not the case with R-Chol. The rate of distribution of R-Chol is similar to that of the free cholesterol. Therefore, R-Chol is the novel fluorescent cholesterol analog that helps to localize the free cholesterol in the cell membrane of the live cells without affecting the cell growth.

Reference: Analytical Biochemistry, Elsevier

Article doi: 10.1016/j.ab.2015.09.003

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