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

<ol>
	<li>Fujimoto, T., Parton, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+just+fat:+the+structure+and+function+of+the+lipid+droplet.">Not just fat: the structure and function of the lipid droplet.</a> Cold Spring Harbor Perspectives in Biology. 3 (3), 004838 (2011).</li><li>Grasselli, E., 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=Models+of+non-alcoholic+fatty+liver+disease+and+potential+translational+value:+The+effects+of+3,5-L-diiodothyronine.">Models of non-alcoholic fatty liver disease and potential translational value: The effects of 3,5-L-diiodothyronine.</a> Annals of Hepatology. 16 (5), 707-719 (2017).</li><li>Herdt, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruminant+adaptation+to+negative+energy+balance:+Influences+on+the+etiology+of+ketosis+and+fatty+liver.">Ruminant adaptation to negative energy balance: Influences on the etiology of ketosis and fatty liver.</a> Veterinary Clinics of North America: Food Animal Practice. 16 (2), 215-230 (2000).</li><li>Pino-de la Fuente, 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=Exercise+regulation+of+hepatic+lipid+droplet+metabolism.">Exercise regulation of hepatic lipid droplet metabolism.</a> Life Sciences. 298, 120522 (2022).</li><li>O'Connor, D., Byrne, A., Berselli, G. B., Long, C., Keyes, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mega-stokes+pyrene+ceramide+conjugates+for+STED+imaging+of+lipid+droplets+in+live+cells.">Mega-stokes pyrene ceramide conjugates for STED imaging of lipid droplets in live cells.</a> Analyst. 144 (5), 1608-1621 (2019).</li><li>Gao, G., 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=Control+of+lipid+droplet+fusion+and+growth+by+CIDE+family+proteins.">Control of lipid droplet fusion and growth by CIDE family proteins.</a> Biochimica et Biophysica Acta (BBA). 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. (171), e62543 (2021).</li><li>Liu, L., 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=Effects+of+nonesterified+fatty+acids+on+the+synthesis+and+assembly+of+very+low+density+lipoprotein+in+bovine+hepatocytes+in+vitro.">Effects of nonesterified fatty acids on the synthesis and assembly of very low density lipoprotein in bovine hepatocytes in vitro.</a> Journal of Dairy Science. 97 (3), 1328-1335 (2014).</li><li>Wang, L., Liu, J. Y., Miao, Z. J., Pan, Q. W., Cao, W. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocyte+lipolysis:+from+molecular+mechanisms+of+regulation+to+disease+and+therapeutics.">Adipocyte lipolysis: from molecular mechanisms of regulation to disease and therapeutics.</a> Biochemical Journal. 477 (5), 985-1008 (2020).</li><li>Gluchowski, N. L., Becuwe, M., Walther, T. C., Farese, R. V. <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+liver+disease:+from+basic+biology+to+clinical+implications.">Lipid droplets and liver disease: from basic biology to clinical implications.</a> Nature Reviews Gastroenterology &amp; Hepatology. 14 (6), 343-355 (2017).</li><li>Meex, R. C. R., Schrauwen, P., Hesselink, M. K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+myocellular+fat+stores:+lipid+droplet+dynamics+in+health+and+disease.">Modulation of myocellular fat stores: lipid droplet dynamics in health and disease.</a> American Journal of Physiology. 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.

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

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

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.

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 ...

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

Research

JoVE Journal

Biology

1.9K 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.

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

Anterior Cervical Discectomy and Fusion in the Ovine Model

Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have failed conservative treatment1,5. Since the operation was first described by Cloward2 and Smith and Robinson6 in 1958, a variety refinements in technique, graft material and implants have been made3. In particular, there is a need for safe osteoinductive agents that could benefit selected patients. The ovine model has been shown to have anatomical, biomechanical, bone density and radiological properties that are similar to the human counterpart, the most similar level being C3/44. It is therefore an ideal model in which preclinical studies can be performed. In particular this methodology may be useful to researchers interested in evaluating different devices and biologics, including stem cells, for potential application in human spinal surgery.

This video demonstrates the technique of anterior cervical discectomy and fusion in the ovine model.

Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...