Name: Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells (Video) | JoVE
Uploaded: 2023-03-10T14:40:00
Description: Watch this Scientific Journal Video about Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells at JoVE.com

This research was jointly supported by the National Natural Science Foundation of China (U1904116).

All procedures were approved and performed in accordance with the ethical standards of the Animal Care Committee of Henan Agricultural University (Henan Province, China).
1. Bovine hepatocyte cell culture
<ol>
	<li>Thaw the primary hepatocyte cells17 and centrifuge 400 x g for 4 min at room temperature. 
	NOTE: The primary hepatocyte cells were cultured and maintained following a previously published report17.</li...

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

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Depending on the pathological states, hepatic LDs undergo tremendous changes in their size and number. LDs are widely present in hepatocyte cells and play a key role in liver health and disease18. The quantity and size of LDs are the basis of the current research on the biogenesis of LDs19. The size and number of LDs for cells and tissues reflect their ability to store and release energy. The dynamic changes of LDs maintain the stability of lipid metabolic activities<sup cl...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>BODIPY 493/503</td>
			<td>invitrogen</td>
			<td>2295015</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Cedar oil</td>
			<td>Solarbio</td>
			<td>C7140</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>cell counting chamber</td>
			<td></td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>cell culture dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>material</td>
		</tr>
		<tr>
			<td>cell sens software&#160;</td>
			<td>Olympus IX73</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30022.01</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>2492319</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>hematoxylin</td>
			<td>DingGuo</td>
			<td>AR0712</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Image view</td>
			<td></td>
			<td></td>
			<td>image analysis sodtware</td>
		</tr>
		<tr>
			<td>linoleic acid</td>
			<td>Solarbio</td>
			<td>SL8520</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Live Cell Station</td>
			<td>Nikon A1 HD25</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>NIS-Elements&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>software</td>
		</tr>
		<tr>
			<td>oil red O</td>
			<td>Solarbio</td>
			<td>G1260</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>optical microscope</td>
			<td>Olympus IX73</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>Penicillin &#38; Streptomycin 100&#215;</td>
			<td>NCM Biotech</td>
			<td>CLOOC5</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>HyClone</td>
			<td>SH30258.01</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>Sealing agent</td>
			<td>Solarbio</td>
			<td>S2150</td>
			<td>reagent</td>
		</tr>
	</tbody>
</table>

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 of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

The staining of cell LDs is shown in Figure 1. The red dots reflect cell LDs, and the blue dots reflect the nuclei. It can be seen that the size and number of LDs in each picture are different under the treatment of LA.
With the increase in LA dosage, the average diameter and number of LDs showed a significantly increasing trend, depending on LA concentration (Figure 2). As shown in Figure 2A, the number ...

Watch this Scientific Journal Video about Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells at JoVE.com

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells (Video) | JoVE 

The present protocol describes how to use oil red O to dye lipid droplets (LDs), calculate the size and number of LDs in a fatty acid-induced fatty hepatocyte model, and use BODIPY 493/503 to observe the process of small LDs fusing into large LDs by live cell imaging.

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 ...