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Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Published: March 10th, 2023



1College of Veterinary Medicine, 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.

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.

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

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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

  1. 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.

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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 .......

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

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


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Name Company Catalog Number Comments
0.25% trypsin Gibco 25200072 reagent
4% paraformaldehyde Solarbio P1110 reagent
BODIPY 493/503 invitrogen 2295015 reagent
Cedar oil Solarbio C7140 reagent
cell counting chamber equipment
cell culture dish Corning 353002 material
cell sens software  Olympus IX73 software
Centrifuge Eppendorf equipment
DMEM HyClone SH30022.01 reagent
Fetal Bovine Serum Gibco 2492319 reagent
hematoxylin DingGuo AR0712 reagent
Image view image analysis sodtware
linoleic acid Solarbio SL8520 reagent
Live Cell Station Nikon A1 HD25 equipment
NIS-Elements  Nikon software
oil red O Solarbio G1260 reagent
optical microscope Olympus IX73 equipment
Penicillin & Streptomycin 100× NCM Biotech CLOOC5 reagent
Phosphate Buffered Saline HyClone SH30258.01 reagent
Pipette Eppendorf equipment
Sealing agent Solarbio S2150 reagent

  1. Fujimoto, T., Parton, R. G. Not just fat: the structure and function of the lipid droplet. Cold Spring Harbor Perspectives in Biology. 3 (3), 004838 (2011).
  2. Grasselli, E., et al. Models of non-alcoholic fatty liver disease and potential translational value: The effects of 3,5-L-diiodothyronine. Annals of Hepatology. 16 (5), 707-719 (2017).
  3. Herdt, T. H. Ruminant adaptation to negative energy balance: Influences on the etiology of ketosis and fatty liver. Veterinary Clinics of North America: Food Animal Practice. 16 (2), 215-230 (2000).
  4. Pino-de la Fuente, F., et al. Exercise regulation of hepatic lipid droplet metabolism. Life Sciences. 298, 120522 (2022).
  5. O'Connor, D., Byrne, A., Berselli, G. B., Long, C., Keyes, T. E. Mega-stokes pyrene ceramide conjugates for STED imaging of lipid droplets in live cells. Analyst. 144 (5), 1608-1621 (2019).
  6. Gao, G., et al. Control of lipid droplet fusion and growth by CIDE family proteins. Biochimica et Biophysica Acta (BBA). Molecular and Cell Biology of Lipids. 1862 (10), 1197-1204 (2017).
  7. Tütüncü Konyar, S. Dynamic changes in insoluble polysaccharides and neutral lipids in the developing anthers of an endangered plant species, Pancratium maritimum. Plant Systematics and Evolution. 304, 397-414 (2018).
  8. Spangenburg, E. E., Pratt, S. J. P., Wohlers, L. M., Lovering, R. M. Use of BODIPY (493/503) to visualize intramuscular lipid droplets in skeletal muscle. Journal of Biomedicine and Biotechnology. 2011, 598358 (2011).
  9. Mehlem, A., Hagberg, C. E., Muhl, L., Eriksson, U., Falkevall, A. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nature Protocols. 8 (6), 1149-1154 (2013).
  10. Diaz, G., Melis, M., Batetta, B., Angius, F., Falchi, A. M. Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio. Micron. 39 (7), 819-824 (2008).
  11. Duan, X., et al. The synthesis of polarity-sensitive fluorescent dyes based on the BODIPY chromophore. Dyes and Pigments. 89 (3), 217-222 (2011).
  12. Rumin, J., et al. The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnology for Biofuels. 8, 42 (2015).
  13. Fam, T. K., Klymchenko, A. S., Collot, M. Recent advances in fluorescent probes for lipid droplets. Materials. 11 (9), 1768 (2018).
  14. Raboisson, D., Mounié, M., Maigné, &. #. 2. 0. 1. ;. Diseases, reproductive performance, and changes in milk production associated with subclinical ketosis in dairy cows: A meta-analysis and review. Journal of Dairy Science. 97 (12), 7547-7563 (2014).
  15. Ospina, P. A., Nydam, D. V., Stokol, T., Overton, T. R. Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States. Journal of Dairy Science. 93 (4), 1596-1603 (2010).
  16. Campos-Espinosa, A., Guzmán, C. A model of experimental steatosis in vitro: hepatocyte cell culture in lipid overload-conditioned medium. Journal of Visualized Experiments. (171), e62543 (2021).
  17. Liu, L., et al. Effects of nonesterified fatty acids on the synthesis and assembly of very low density lipoprotein in bovine hepatocytes in vitro. Journal of Dairy Science. 97 (3), 1328-1335 (2014).
  18. Wang, L., Liu, J. Y., Miao, Z. J., Pan, Q. W., Cao, W. L. Lipid droplets and their interactions with other organelles in liver diseases. The International Journal of Biochemistry & Cell Biology. 133, 105937 (2021).
  19. Sanjabi, B., et al. Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes. Scientific Reports. 5, 8816 (2015).
  20. Saponaro, C., Gaggini, M., Carli, F., Gastaldelli, A. The subtle balance between lipolysis and lipogenesis: a critical point in metabolic homeostasis. Nutrients. 7 (11), 9453-9474 (2015).
  21. Yang, A., Mottillo, E. P. Adipocyte lipolysis: from molecular mechanisms of regulation to disease and therapeutics. Biochemical Journal. 477 (5), 985-1008 (2020).
  22. Gluchowski, N. L., Becuwe, M., Walther, T. C., Farese, R. V. Lipid droplets and liver disease: from basic biology to clinical implications. Nature Reviews Gastroenterology & Hepatology. 14 (6), 343-355 (2017).
  23. Meex, R. C. R., Schrauwen, P., Hesselink, M. K. C. Modulation of myocellular fat stores: lipid droplet dynamics in health and disease. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 297 (4), 913-924 (2009).
  24. Sarnyai, F., et al. Effect of cis-and trans-monounsaturated fatty acids on palmitate toxicity and on palmitate-induced accumulation of ceramides and diglycerides. International Journal of Molecular Sciences. 21 (7), 2626 (2020).
  25. Ricchi, M., et al. Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. Journal of Gastroenterology and Hepatology. 24 (5), 830-840 (2009).
  26. Fei, W., et al. A role for phosphatidic acid in the formation of "supersized" lipid droplets. PLoS Genetics. 7 (7), e1002201 (2011).
  27. Kowada, T., Maeda, H., Kikuchi, K. BODIPY-based probes for the fluorescence imaging of biomolecules in living cells. Chemical Society Reviews. 44 (14), 4953-4972 (2015).
  28. Wang, J., et al. Application of the fluorescent dye BODIPY in the study of lipid dynamics of the rice blast fungus Magnaporthe oryzae. Molecules. 23 (7), 1594 (2018).

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