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>
	<li>Discard the frozen storage solution with a pipette and suspend it with 1 mL of medium containing 10% fetal bovine serum (FBS) and Dulbecco&#39;s modified Eagle&#39;s medium (DMEM). Next, use a cell counting chamber to calculate the number of cells per milliliter, and then calculate the concentration of the above cell suspension and adjust the cell concentration to 1 x 107 cells/mL.
	<ol>
		<li>Add the cell suspension into a 60 mm cell culture dish containing 3 mL of the above medium. Culture the cells and incubate them at 37 &#176;C and 5% CO2 for 24 h.</li>
	</ol>
	</li>
	<li>When the cells grow to 80%, discard the medium and rinse with phosphate-buffered saline (PBS), then add 750 &#181;L of 0.25% trypsin to digest the cells. Incubate the cells at 37 &#176;C for 3 min, then neutralize them with the same amount of 10% FBS. Collect the cell suspension to centrifuge at 200 x g for 4 min at room temperature.</li>
</ol>
2. Oil red O staining
<ol>
	<li>Add 800 &#181;L of culture medium containing 10% FBS and DMEM into each well of the 24-well cell culture plate containing glass coverslips. Take 50 &#181;L of the cell suspension (obtained in step 1.3) and add it to 950 &#181;L of PBS to mix. Use a cell counting chamber to calculate the number of cells per milliliter, and then calculate the concentration of the above cell suspension. Finally, adjust the cell concentration to 4 x 104/mL in each well and incubate at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>After 24 h, discard the culture medium and wash with PBS. Suspend with 800 &#181;L of DMEM induction medium containing 1 mg/mL bovine serum albumin (BSA).</li>
	<li>Dissolve a total of 100 &#181;L of LA (see Table of Materials) in 900 &#181;L of anhydrous ethanol and prepare a standard solution (100 mmol/L). Add a gradient of 0 &#181;mol/L, 100 &#181;mol/L, 150 &#181;mol/L, and 200 &#181;mol/L LA into the 24-well plate. Repeat each treatment four times. Incubate at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Remove the culture medium and wash the cells with PBS three times. Fix with 400 &#181;L of 4% paraformaldehyde for 20 min. Discard the fixative solution and wash it with PBS three times.</li>
	<li>Incubate the cells with 60% isopropyl alcohol for 5 min, then discard it. Add freshly prepared oil red O working solution (see Table of Materials) (3:2 ratio of oil red O:water) for 20-30 min and discard the staining solution. Wash the cells with PBS two to five times until there is no excess dye solution.</li>
	<li>Add 300 &#181;L of hematoxylin staining solution (hematoxylin:water ratio of 1:10) and re-dye the nucleus for 1-2 min. Discard the dye solution and wash the cells with PBS two to five times. Take out the glass coverslips from the 24-well plate and place them on microscopic slides (the side with cells facing down) after dropping 10 &#181;L of tablet sealant (see Table of Materials) onto the slide.</li>
	<li>After sealing, observe and image the LDs of the cells under the oil lens of the optical microscope (see Table of Materials). Measure the diameter of the LDs by cellSens software and analyze the number and size of the LDs.</li>
</ol>
3. Measurement of the size and number of LDs
<ol>
	<li>Capture images: Turn on the computer and microscope switch successively, place the slide on the loading platform, open the image analysis software (see Table of Materials), and connect the computer to view the image.
	<ol>
		<li>Find the images at low power and drip an appropriate amount of cedar oil on the imaging slide. Adjust the observation factor to 100x, set automatic exposure, and capture the images by adjusting the appropriate field of view. Select three stained cell slides for each group for imaging.</li>
	</ol>
	</li>
	<li>Diameter measurement: Randomly select 60 LDs for each image to measure the diameters. After the measurement of each image, save the images and output the measurement results into a table for the subsequent analysis of the average size and distribution ratio of LDs.</li>
	<li>Quantity measurement: Select three images for each stained and photographed slide, and randomly select three cells for quantity measurement in each picture. Count and analyze the number of LDs around the cell, and calculate the average number of LDs in the cell.</li>
</ol>
4. Dynamic observation of LD fusion
<ol>
	<li>Follow the cell culture steps as mentioned in steps 1.1-1.3. When the cells grow to 80%, discard the medium and rinse with PBS, then digest them with 750 &#181;L of trypsin for 3 min. Next, add 750 &#956;L of the medium, centrifuge at 200 x g for 4 min at room temperature, and discard the supernatant.</li>
	<li>Suspend the cells in 1 mL of culture medium, count, and adjust the cell concentration to 5 x 105 cells/mL in a 35 mm dish. Culture at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>When the cells have grown to 80%, change the medium to DMEM + 150 &#181;mol/L LA for 24 h, and continue the culture in an incubator at 37 &#176;C and 5% CO2 to accumulate the LDs.</li>
	<li>After 24 h, remove the culture medium. Wash the adherent cells with PBS and incubate them with 10 &#181;g/mL BODIPY 493/503 neutral fluorescent probe (see Table of Materials) in the dark for 30 min. After incubation, wash the cells in the culture dish with PBS three times and add DMEM + 150 &#181;mol/L LA. 
	NOTE: Avoid light exposure during and after the 10 &#181;g/mL BODIPY staining; 1 mg/mL BODIPY was diluted with PBS (1:100).</li>
	<li>Place the culture dish in the groove of the microscope of the living cell station (see Table of Materials) to observe the dynamic changes of LDs. Turn on the power of the living cell workstation according to the starting sequence and avoid light.
	<ol>
		<li>Turn on the power, transmission light source, microscope power source, mercury lamp fluorescent light source, charge-coupled device (CCD) camera power source, computer host power source, CO2 valve, and CO2 incubator.</li>
	</ol>
	</li>
	<li>Add distilled water to the groove on the loading platform, ensuring not to go over the air vent.</li>
	<li>Turn on the computer and run &#34;NIS-Elements&#34;. First, find the appropriate field of view on the 4x objective lens, then adjust it to the 40x objective lens successively. Select E100 for observing the sample in the microscope and L100 for previewing and photographing the sample on the computer. 
	NOTE: E100 and L100 are microscope adjustment buttons on the living cell workstation (see Table of Materials), representing the eyepiece and the computer screen, respectively.</li>
	<li>Design the parameters such as fluorescence channel (e.g., Ph-40x, Fluorescein isothiocyanate [FITC], shutter) and expected shooting time for the experiment. Set the shooting time in time, including the shooting interval time of 5 min between every two images and a total shooting time of 6 h. 
	NOTE: Long-time shooting needs to use the perfect focus system (PFS) focus stabilization function. For this, first adjust the field of vision and the focus length, then click on PFS on the computer screen, and finally adjust the fine focus spiral.</li>
	<li>Select different channel modes, single channel or all, select a different field of view, click preview, adjust the field of view, set these parameters, and click on start running to start shooting. Take a test shot for 5 min first; it can take a long time after the operation is normal.</li>
	<li>After the shooting is performed, choose File &#62; Save as &#62; Save type &#62; avi format to export the data to a video in &#39;avi&#39; format. Use the &#39;.nd2&#39; image format to save the data program and export the photos.</li>
	<li>For powering off the machine, turn it off in reverse order.</li>
</ol>
5. Statistics and result analysis
<ol>
	<li>Analyze the data using one-way ANOVA. Report the results as the mean &#177; standard error.</li>
</ol>

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

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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.

1.1K 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

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