Abstract
Lipid bodies (LB) are dynamic inducible organelles with the key roles in cellular lipid metabolism, intracellular trafficking and signaling. These structures have a neutral lipid-rich core which contains mainly triacylglycerides (TAG) and cholesterol esters (CE). With the use of flow cytometry and lipophylic fluorescent dye Nile red (NR) we studied LB biogenesis in nonfixed freshly isolated epithelial cells derived from the frog (Rana temporaria L.) urinary bladder. These cells are characterized by numerous small LB located diffusely in the cytoplasm. To target neutral lipids in LB, we used arachidonic acid (AA), an inducer of LB biogenesis in different cell types, and methyl-β-cyclodextrin (MβCD), non-permeable cholesterol acceptor, widely used to extract cholesterol from the lipid rafts. The cells were incubated with 10–50 μM AA for 1 h or with 400–2000 μM MβCD for 30 min; after that they were stained with NR, and fluorescence was measured by flow cytometer at λex = 488 nm and λem = 575 ± 15 nm. In parallel, side scatter (SS) was analyzed. It was found that AA in a dose-dependent manner increased NR fluorescence. At a maximal concentration used, AA increased NR fluorescence and SS by 41 ± 2% and by 15 ± 3% (p < 0.001), respectively. Analysis of lipid composition of cell extracts revealed a significant increase of TAG by 10 μM AA. MβCD starting from 400 μM decreased dose-dependently the NR fluorescence and SS. Its effect was accompanied by a decrease of cellular free cholesterol by 6% (p < 0.01) and cholesterol ester by 21% (p < 0.001). This fact indicates mobilization of cholesterol from cholesterol esters stored in LB in order to restore cholesterol level in the plasma membrane. Taken together, our data demonstrate that flow cytometry in combination with NR staining represents a reliable tool to be used for recording of changes in different neutral lipids class content within LB in living cells non-specialized on the fat storage.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
