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

<ol>
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Molecular and Cell Biology of Lipids. 1862 (10), 1197-1204 (2017).</li><li>T&#252;t&#252;nc&#252; Konyar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+insoluble+polysaccharides+and+neutral+lipids+in+the+developing+anthers+of+an+endangered+plant+species,+Pancratium+maritimum.">Dynamic changes in insoluble polysaccharides and neutral lipids in the developing anthers of an endangered plant species, Pancratium maritimum.</a> Plant Systematics and Evolution. 304, 397-414 (2018).</li><li>Spangenburg, E. E., Pratt, S. J. P., Wohlers, L. M., Lovering, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+BODIPY+(493/503)+to+visualize+intramuscular+lipid+droplets+in+skeletal+muscle.">Use of BODIPY (493/503) to visualize intramuscular lipid droplets in skeletal muscle.</a> Journal of Biomedicine and Biotechnology. 2011, 598358 (2011).</li><li>Mehlem, A., Hagberg, C. E., Muhl, L., Eriksson, U., Falkevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+neutral+lipids+by+oil+red+O+for+analyzing+the+metabolic+status+in+health+and+disease.">Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease.</a> Nature Protocols. 8 (6), 1149-1154 (2013).</li><li>Diaz, G., Melis, M., Batetta, B., Angius, F., Falchi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+characterization+of+intracellular+lipids+in+situ+by+Nile+Red+red/yellow+emission+ratio.">Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio.</a> Micron. 39 (7), 819-824 (2008).</li><li>Duan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+of+polarity-sensitive+fluorescent+dyes+based+on+the+BODIPY+chromophore.">The synthesis of polarity-sensitive fluorescent dyes based on the BODIPY chromophore.</a> Dyes and Pigments. 89 (3), 217-222 (2011).</li><li>Rumin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+Nile+red+and+BODIPY+for+lipid+measurement+in+microalgae.">The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae.</a> Biotechnology for Biofuels. 8, 42 (2015).</li><li>Fam, T. K., Klymchenko, A. S., Collot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+fluorescent+probes+for+lipid+droplets.">Recent advances in fluorescent probes for lipid droplets.</a> Materials. 11 (9), 1768 (2018).</li><li>Raboisson, D., Mouni&#233;, M., Maign&#233;, &. #. 2. 0. 1. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases,+reproductive+performance,+and+changes+in+milk+production+associated+with+subclinical+ketosis+in+dairy+cows:+A+meta-analysis+and+review.">Diseases, reproductive performance, and changes in milk production associated with subclinical ketosis in dairy cows: A meta-analysis and review.</a> Journal of Dairy Science. 97 (12), 7547-7563 (2014).</li><li>Ospina, P. A., Nydam, D. V., Stokol, T., Overton, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+of+elevated+nonesterified+fatty+acids+and+&#946;-hydroxybutyrate+concentrations+with+early+lactation+reproductive+performance+and+milk+production+in+transition+dairy+cattle+in+the+northeastern+United+States.">Associations of elevated nonesterified fatty acids and &#946;-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.</a> Journal of Dairy Science. 93 (4), 1596-1603 (2010).</li><li>Campos-Espinosa, A., Guzm&#225;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+experimental+steatosis+in+vitro:+hepatocyte+cell+culture+in+lipid+overload-conditioned+medium.">A model of experimental steatosis in vitro: hepatocyte cell culture in lipid overload-conditioned medium.</a> Journal of Visualized Experiments. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+and+their+interactions+with+other+organelles+in+liver+diseases.">Lipid droplets and their interactions with other organelles in liver diseases.</a> The International Journal of Biochemistry &amp; Cell Biology. 133, 105937 (2021).</li><li>Sanjabi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+hypertrophy:+a+crucial+determining+factor+in+insulin+regulation+by+adipocytes.">Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes.</a> Scientific Reports. 5, 8816 (2015).</li><li>Saponaro, C., Gaggini, M., Carli, F., Gastaldelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subtle+balance+between+lipolysis+and+lipogenesis:+a+critical+point+in+metabolic+homeostasis.">The subtle balance between lipolysis and lipogenesis: a critical point in metabolic homeostasis.</a> Nutrients. 7 (11), 9453-9474 (2015).</li><li>Yang, A., Mottillo, E. 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Regulatory, Integrative and Comparative Physiology. 297 (4), 913-924 (2009).</li><li>Sarnyai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cis-and+trans-monounsaturated+fatty+acids+on+palmitate+toxicity+and+on+palmitate-induced+accumulation+of+ceramides+and+diglycerides.">Effect of cis-and trans-monounsaturated fatty acids on palmitate toxicity and on palmitate-induced accumulation of ceramides and diglycerides.</a> International Journal of Molecular Sciences. 21 (7), 2626 (2020).</li><li>Ricchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effect+of+oleic+and+palmitic+acid+on+lipid+accumulation+and+apoptosis+in+cultured+hepatocytes.">Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes.</a> Journal of Gastroenterology and Hepatology. 24 (5), 830-840 (2009).</li><li>Fei, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+phosphatidic+acid+in+the+formation+of+&amp;#34;supersized&amp;#34;+lipid+droplets.">A role for phosphatidic acid in the formation of &amp;#34;supersized&amp;#34; lipid droplets.</a> PLoS Genetics. 7 (7), e1002201 (2011).</li><li>Kowada, T., Maeda, H., Kikuchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BODIPY-based+probes+for+the+fluorescence+imaging+of+biomolecules+in+living+cells.">BODIPY-based probes for the fluorescence imaging of biomolecules in living cells.</a> Chemical Society Reviews. 44 (14), 4953-4972 (2015).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+fluorescent+dye+BODIPY+in+the+study+of+lipid+dynamics+of+the+rice+blast+fungus+Magnaporthe+oryzae.">Application of the fluorescent dye BODIPY in the study of lipid dynamics of the rice blast fungus Magnaporthe oryzae.</a> Molecules. 23 (7), 1594 (2018).</li></ol>

The authors declare that they have no conflicts of interest.

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.

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

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

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.

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

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 ...

evaluation-of-lipid-droplet-size-and-fusion-in-bovine-hepatic-cells

Research

JoVE Journal

Biology

Oil Red O Staining and Measurement of Lipid Droplets (LDs) in Bovine Hepatocyte Cells

To begin, take a previously seated 24 well culture plate and place a glass cover slip in each well. Then add 800 microliters of culture medium containing 10%FBS and DMEM. After adjusting cell concentration.Incubate it at 37 degrees Celsius and 5%carbon dioxide for 24 hours. Then discard the culture medium and wash the cells with PBS. Suspend the cells in 800 microliters of DMEM induction medium containing BSA.Dissolve linoleic acid in anhydrous ethanol to prepare a standard solution at a concentration of 100 millimolar per liter. Add linoleic acid in increasing gradient to the plate and repeat it four times. Then incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 24 hours.At the end of the incubation, remove the culture medium and wash the cells with PBS three times. Fix them with 400 microliters of 4%paraform aldehyde for 20 minutes. Then discard the fixative solution and wash them again.Incubate the cells with 60%isopropyl alcohol for five minutes then discard it. Add freshly prepared oil red O working solution and discard it after 20 to 30 minutes. Wash the cells with PBS two to five times until the excess dye solution is removed.Restain the nucleus with 300 microliters of hematoxylin solution for one to two minutes. Then discard the dye solution and wash them again. Remove the glass cover slip from the PBS washed plate and place it with cells facing down on a microscopic slide containing 10 microliters of tablet sealant.To measure the size and number of LDs, turn on the computer and microscope switch successively and place the slide on the loading platform. Then open the image analysis software. Find the images at low power and drip an appropriate amount of cedar oil on the imaging slide.Gradually adjust the objective 200x and adjust the field of view until a clear image is found. Then capture images from the desired regions and save them. Randomly, select 60 LDs for each image and measure the diameter.Export the measurement results into a table to analyze LDs average size and distribution ratio. The stained hepatocyte cultured cells showed different sizes and numbers of LDs. Under the different concentrations of linoleic acid treatment.The number of LDs per cell was negatively correlated with different concentrations of linoleic acid. The median LD number per cell was 136 for the 100 micromolar per liter linoleic acid treated group and decreased at high concentrations. The average diameter of LDs in the control group was 0.72 micrometers which increased with increasing concentration in the linoleic acid treated group.A high linoleic acid concentration increased the proportion of large LDs and decreased small LDs.

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

To begin, obtain 80%grown bovine hepatocyte cells and replace the culture medium with DMEM containing linoleic acid. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide for 24 hours to accumulate the LDs. Then remove the culture medium and wash the adherence cells with PBS.Add BODIPY neutral fluorescent probe in the cells and incubate in the dark for 30 minutes. After three PBS washes, add DMEM containing linoleic acid to the cells. Place the culture dish in the groove of the microscope of the living cell station and turn on the microscope and computer.Run NIS-Elements software. Find the field of view at 4x magnification. Then adjust it to the 40x, turn PFS on, refocus, and select the appropriate field of view.For the test run, set the appropriate time and channels. Then press Run Now to shoot the samples for one minute. For the experiment, in the Time tab, set the interval time of five minutes and shoot duration of six hours, set fluorescent and bright-field channels, and press Run now to shoot the samples.Then choose File followed by Save As and AVI Image File format to export the data to a video in AVI format. Large LDs were formed through the fusion of small LDs. In the first 15 minutes, there were smaller LDs.From 20 minutes, they started to fuse and were completely fused into larger LDS by 35 minutes.

1.0K Views.  Henan Agricultural University.  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.

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

Summary