Name: Dynamic Observation of Lipid Droplet (LD) Fusion in Bovine Hepatocyte Cells
Uploaded: 2023-03-10T14:40:00
Description: Watch this Scientific Journal Video about Dynamic Observation of Lipid Droplet LD Fusion in Bovine Hepatocyte Cells at JoVE.com

dynamic observation of lipid droplet (ld) fusion in bovine hepatocyte cells

Live Cell Imaging of Lipid Droplet Size and Fusion Using Oil Red O and BODIPY

Lipid droplet (LD) accumulation in hepatocytes is the typical characteristic of non-alcoholic fatty liver disease (NAFLD), which can progress to liver fibrosis and hepatocellular carcinoma. It has been found that the earliest manifestation of fatty liver disease is steatosis, characterized by LD accumulation in the cytoplasm of the hepatocyte1. Liver steatosis is invariably associated with an increased number and/or expanded size of LDs2. LDs are thought to be generated from the endoplasmic reticulum (ER), consisting of triglyceride (TG) as the core, and are surrounded by proteins and phospholipids3. As the subcellular organelle responsible for TG storage, LDs exhibit different features regarding their size, number, lipid composition, proteins, and interaction with other organelles, all of which affect cell energy homeostasis4. The TG level is positively correlated with the size of LDs, and a higher intracellular TG content could form larger LDs5. LDs increase in size through the local synthesis of TG, lipid incorporation in the ER, and the fusion of multiple LDs6. Cells (adipocytes, hepatocytes, etc.) that contain large LDs have a special mechanism to efficiently increase lipid storage by LD fusion. The dynamic changes of LDs reflect the different energy metabolism states of the cell. It is crucial to develop methodologies that allow the observation and analysis of the various hepatic LDs in healthy and abnormal cells.
The main non-fluorescent dyes for LDs are Sudan Black B and oil red O. Sudan Black B stains neutral lipids, phospholipids, and steroids7. Oil red O is mainly used for staining LDs of skeletal muscle, cardiomyocytes, liver tissue, adipose cells, etc8., and is considered a standard tool for the quantitative detection of liver steatosis in mice and humans9. The dynamic change of LDs is mainly carried out by fluorescence dyeing. Nile red and BODIPY are both commonly used fluorescent lipid dyes10,11. Compared with Nile red, BODIPY has stronger tissue permeability and binds better with LDs12. BODIPY-labeled LDs can be used for staining living cells and colocalization with other organelles13.
The incidence of fatty liver disease is significantly higher in ruminant animals than in monogastric animals14. During the transition period, dairy cows experience a state of negative energy balance3. Large quantities of non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid, etc.) are synthesized into TGs in bovine hepatocytes, which leads to liver functional abnormality and greatly reduces the quality of milk products and production efficiency15. The present study aims to provide a protocol to analyze the size and the number of LDs, as well as to monitor the LD fusion dynamics. We constructed a model of LD formation by adding different concentrations of linoleic acid (LA) in hepatocytes16 and observed the changes in the size and the number of LDs during the process by staining LDs with oil red O. In addition, the process of the rapid fusion of LDs was also observed by staining with BODIPY 493/503.

Author Spotlight: Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells  

Utvärdering av lipiddroppstorlek och fusion i bovina leverceller

Our research focus on the lipid droplet number and the size induced by linoleic acid in liver cells. Additionally, we aim to provide insights into the mechanism of the fatty liver. Recent advancements in this field of research, suggests that choline, CCT alpha, established induced autophagy, can regulate lipid droplet size in bovine hepatic cells.Moreover, living cell workstation observations have shown that phospholipids have a considerable effect on lipid droplet fusion. Currently, gene aiding, fluorescence tweezer, liquid chromatography, omics analysis, single chromatography, flow cytometry, and other technologies are being used to group the vast research in this field. The experimental method has some limitations.For example, the phenomenon of limited fields can only be observed for lipid droplet fields and under a certain flight of waste. Oil Red O staining is more convenient, less expensive, and more widely used to measure the apparent size of lipid droplets than lipid droplet fluorescence staining. It is simple to operate and requires less equipment.Furthermore, we directly observe this phenomenal of lipid fusion through the living cell station. This study provide a simple and repeatable method to observe lipid droplet size and fusion, which can be widely applied to lipid droplet analysis in cellular lipid metabolism research. We will continue to conduct intensive research on lipid droplets, focusing on the mechanism of lipid droplet fusion and the structure changes of fatty liver in dairy cows.

Watch this Scientific Journal Video about Dynamic Observation of Lipid Droplet LD Fusion in Bovine Hepatocyte Cells at JoVE.com

Dynamic Observation of Lipid Droplet (LD) Fusion in Bovine Hepatocyte Cells  

Dynamisk observation av lipiddroppfusion (LD) i bovina hepatocytceller

För att börja, få 80% odlade bovina hepatocytceller och ersätt odlingsmediet med DMEM innehållande linolsyra. Inkubera cellerna vid 37 grader Celsius i 5% koldioxid i 24 timmar för att ackumulera LD. Ta sedan bort odlingsmediet och tvätta vidhäftningscellerna med PBS.Tillsätt BODIPY neutral fluorescerande sond i cellerna och inkubera i mörker i 30 minuter. Efter tre PBS-tvättar, tillsätt DMEM innehållande linolsyra till cellerna. Placera odlingsskålen i spåret på mikroskopet på den levande cellstationen och sätt på mikroskopet och datorn.Kör NIS-Elements-programvaran. Hitta synfältet vid 4x förstoring. Justera den sedan till 40x, slå på PFS, fokusera om och välj lämpligt synfält.För testkörningen ställer du in lämplig tid och kanaler. Tryck sedan på Kör nu för att ta proverna i en minut. För experimentet ställer du in intervalltiden på fem minuter på fliken Tid och inspelningstiden på sex timmar, ställer in fluorescerande kanaler och ljusfältskanaler och trycker på Kör nu för att ta proverna.Välj sedan Fil följd av Spara som och AVI Image File format för att exportera data till en video i AVI-format. Stora LD bildades genom fusion av små LD. Under de första 15 minuterna fanns det mindre LD.Efter 20 minuter började de smälta samman och var helt sammansmälta till större LDS med 35 minuter.

Research

JoVE Journal

Biology

Dynamic Observation of Lipid Droplet (LD) Fusion in Bovine Hepatocyte Cells

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...