Application of flow cytometry for estimation of lipid content changes induced by arachidonic acid and methyl-β-cyclodextrin in the lipid bodies of epithelial cells



Lipid bodies (LB) are dynamic inducible organelles with the key roles in cellular lipid metabolism, intracellular trafficking and signaling. These structures have a neutral lipid-rich core which contains mainly triacylglycerides (TAG) and cholesterol esters (CE). With the use of flow cytometry and lipophylic fluorescent dye Nile red (NR) we studied LB biogenesis in nonfixed freshly isolated epithelial cells derived from the frog (Rana temporaria L.) urinary bladder. These cells are characterized by numerous small LB located diffusely in the cytoplasm. To target neutral lipids in LB, we used arachidonic acid (AA), an inducer of LB biogenesis in different cell types, and methyl-β-cyclodextrin (MβCD), non-permeable cholesterol acceptor, widely used to extract cholesterol from the lipid rafts. The cells were incubated with 10–50 μM AA for 1 h or with 400–2000 μM MβCD for 30 min; after that they were stained with NR, and fluorescence was measured by flow cytometer at λex = 488 nm and λem = 575 ± 15 nm. In parallel, side scatter (SS) was analyzed. It was found that AA in a dose-dependent manner increased NR fluorescence. At a maximal concentration used, AA increased NR fluorescence and SS by 41 ± 2% and by 15 ± 3% (p < 0.001), respectively. Analysis of lipid composition of cell extracts revealed a significant increase of TAG by 10 μM AA. MβCD starting from 400 μM decreased dose-dependently the NR fluorescence and SS. Its effect was accompanied by a decrease of cellular free cholesterol by 6% (p < 0.01) and cholesterol ester by 21% (p < 0.001). This fact indicates mobilization of cholesterol from cholesterol esters stored in LB in order to restore cholesterol level in the plasma membrane. Taken together, our data demonstrate that flow cytometry in combination with NR staining represents a reliable tool to be used for recording of changes in different neutral lipids class content within LB in living cells non-specialized on the fat storage.


lipid bodies flow cytometer arachidonic acid methyl-β-cyclodextrin triacylglycerol cholesterol cholesterol esters epithelial cells frog urinary bladder 


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  1. 1.
    Fujimoto T., Ohsaki Y., Cheng J., Suzuki M., Shinohara Y. 2008. Lipid droplets: A classic organelle with new outfits. Histochem. Cell. Biol. 130(2), 263–279.PubMedCrossRefGoogle Scholar
  2. 2.
    Ohsaki Y., Cheng J., Suzuki M., Shinohara Y., Fujita A., Fujimoto T. 2008. Biogenesis of cytoplasmic lipid droplets: From the lipid ester globule in the membrane to the visible structure. Biochim. Biophys. Acta. 1791, 399–407.PubMedGoogle Scholar
  3. 3.
    Greenberg A., Egan J., Wek S.A., Garty N., Blanchette-Mackie E., Londos C. 1991. Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. J. Biol. Chem. 266(17), 11341–11346.PubMedGoogle Scholar
  4. 4.
    Wolins N., Rubin B., Brasaemle D. 2001. TIP47 associates with lipid droplets. J. Biol. Chem. 276, 5101–5108.PubMedCrossRefGoogle Scholar
  5. 5.
    Londos C., Sztalryd C., Tansey J., Kimmel A. 2005. Role of PAT proteins in lipid metabolism. Biochimie. 87(1), 45–49.PubMedCrossRefGoogle Scholar
  6. 6.
    Brasaemle D. 2007. Thematic review series: Adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis. J. Lipid Res. 48(12), 2547–2559.PubMedCrossRefGoogle Scholar
  7. 7.
    Johnson M., Vaughn B., Triggiani M., Swan D., Fonteh A., Chilton F. 1999. Role of arachidonyl triglycerides within lipid bodies in eicosanoid formation by human polymorphonuclear cells. Am. J. Respire Cell. Mol. Biol. 21, 253–258.CrossRefGoogle Scholar
  8. 8.
    Farese R., Walther T. 2009. Lipid droplets finally get a little R-E-S-P-E-C-T. Cell. 139, 855–860.PubMedCrossRefGoogle Scholar
  9. 9.
    Olofsson S., Bostrom P., Andersson L. et al., 2009. Lipid droplets as dynamic organelles connecting storage and efflux of lipids. Biochim. Biophys. Acta. 1791(6), 448–458.PubMedCrossRefGoogle Scholar
  10. 10.
    Walther T., Farese R. 2009. The life of lipid droplets. Biochim. Biophys. Acta. 1791(6), 459–466.PubMedCrossRefGoogle Scholar
  11. 11.
    Roingeard P., Hourioux C. 2008. Hepatitis C virus core protein, lipid droplets and steatosis. J. Virus Hepatitis. 15, 157–164.CrossRefGoogle Scholar
  12. 12.
    Bozza P.T., Yu W., Penrose J.F., Morgan E.S., Dvorak A.M., Weller P.F. 1997. Eosinophils lipid bodies: Specific, inducible intracellular sites for enhanced eicosanoid formation. J. Exp. Med. 186, 909–920.PubMedCrossRefGoogle Scholar
  13. 13.
    Bozza P., Viola J. 2010. Lipid droplets in inflammation and cancer. Prostaglandins, Leucotrienes and Essential Fatty Acids. 82, 243–250.CrossRefGoogle Scholar
  14. 14.
    Dvorak A.M., Morgan E., Tzizik D.M., Weller P.F. 1994. Prostaglandin endoperoxide synthase (cyclooxygenase): Ultrastructural localization to non-membrane-bound cytoplasmic lipid bodies in human eosinophils and murine 3T3 fibroblasts. Int. Arch. Allergy Immunol. 105, 245–250.PubMedCrossRefGoogle Scholar
  15. 15.
    Maya-Monteiro C.M., Almeida P.E., D’Avila H., Martins A.S., Rezende A.P., Castro-Faria-Neto H., Bozza P.T. 2008. Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism. J. Biol. Chem. 283(4), 2203–2210.PubMedCrossRefGoogle Scholar
  16. 16.
    Weller P.F., Ryeom S.W., Picard S.T., Ackerman S.J., Dvorak A.M. 1991. Cytoplasmic lipid bodies of neutrophils: Formation induced by cis-unsaturated fatty acids and mediated by protein kinase C. J. Cell Biol. 113(1), 137–146.PubMedCrossRefGoogle Scholar
  17. 17.
    Moreira L.S., Piva B., Gentile L.B., Mesquita-Santos F.P., D’Avila H., Maya-Monteiro C.M., Bozza P.T., Bandeira-Melo C., Diaz B.L. 2009. Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells. Biochim. Biophys. Acta. 1791(3), 156–165.PubMedCrossRefGoogle Scholar
  18. 18.
    Bozaquel-Morais B., Madeira J., Maya-Monteiro C., Masuda C., Montero-Lomeli M. 2010. A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism. PLoS One. 28, 5 (10), doi: 10.1371/journal.pone.0013692.Google Scholar
  19. 19.
    Greenspan P., Mayer E.P., Fowler S.D. 1985. Nile red: A selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100(3), 965–973.PubMedCrossRefGoogle Scholar
  20. 20.
    Schaedlich K., Knelangen J.M, Navarrete Santos A., Fischer B., Navarrete Santos A. 2010. A simple method to sort ESC-derived adipocytes. Cytometry A. 77(10), 990–995.PubMedGoogle Scholar
  21. 21.
    Hassall D.G. 1992. Three probe flow cytometry of a human foam-cell forming macrophage. Cytometry. 13(4), 381–388.PubMedCrossRefGoogle Scholar
  22. 22.
    da Silva T.L., Feijão D., Reis A. 2010. Using multiparameter flow cytometry to monitor the yeast Rhodotorula glutinis CCMI 145 batch growth and oil production towards biodiesel. Appl. Biochem. Biotechnol. 162(8), 2166–2176.PubMedCrossRefGoogle Scholar
  23. 23.
    Raschke D., Knorr D. 2009. Rapid monitoring of cell size, vitality and lipid droplet development in the oleaginous yeast Waltomyces lipofer. J. Microbiol. Methods. 79(2), 178–183.PubMedCrossRefGoogle Scholar
  24. 24.
    Cirulis J.T., Strasser B.C., Scott J.A., Ros G.M. 2012. Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability. Cytometry A. 81, 618–626.PubMedGoogle Scholar
  25. 25.
    Greenspan P., Fowler S.D. 1985. Spectrofluorometric studies of the lipid probe, nile red. J. Lipid Res. 26(7), 781–789.PubMedGoogle Scholar
  26. 26.
    Triggiani M., Oriente A., de Crescenzo G., Rossi G., Marone G. 1995. Biochemical functions of a pool of arachidonic acid associated with triglycerides in human inflammatory cells. Int. Arch. Allergy Immunol. 107(1–3), 261–263.PubMedCrossRefGoogle Scholar
  27. 27.
    Dichlberger A., Schlager S., Lappalainen J., Käkelä R., Hattula K., Butcher S.J., Schneider W.J., Kovanen P.T. 2011. Lipid body formation during maturation of human mast cells. J. Lipid Res. 52(12), 2198–2208.PubMedCrossRefGoogle Scholar
  28. 28.
    Zidovetzki R., Levitan I. 2007. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim. Biophys. Acta. 1768(6), 1311–1124.PubMedCrossRefGoogle Scholar
  29. 29.
    Chang T., Chang C., Ohgami N., Yamauchi Y. 2006. Cholesterol sensing, trafficking, and esterification. Annu. Rev. Cell Dev. Biol. 22, 129–157.PubMedCrossRefGoogle Scholar
  30. 30.
    Mahammad S., Parmryd I. 2008. Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions. Biochim. Biophys. Acta. 1778(5), 1251–1258.PubMedCrossRefGoogle Scholar
  31. 31.
    Folch J., Lees M., Sloane-Stanley G. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509.PubMedGoogle Scholar
  32. 32.
    Gubern A., Casas J., Barceló-Torns M., Barneda D., de la Rosa X., Masgrau R., Picatoste F., Balsinde J., Balboa M.A., Claro E. 2008. Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets. J. Biol. Chem. 283(41), 27369–27382.PubMedCrossRefGoogle Scholar
  33. 33.
    Diaz G., Melis M., Batetta B., Angius F., Falchi A. 2008. Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio. Micron. 39(7), 819–824.PubMedCrossRefGoogle Scholar
  34. 34.
    Romek M., Gajda B., Krzysztofowicz E., Kepczynski M., Smorag Z. 2011. New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe. Theriogenology. 75(1), 42–54.PubMedCrossRefGoogle Scholar
  35. 35.
    Neufeld E.B., Cooney A.M., Pitha J., Dawidowicz E.A., Dwyer N.K., Pentchev P.G., Blanchette-Mackie E.J. 1996. Intracellular trafficking of cholesterol monitored with a cyclodextrin. J. Biol. Chem. 271, 21604–21613.PubMedCrossRefGoogle Scholar

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© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  1. 1.Sechenov Institute of Evolutionary Physiology and BiochemistryRussian Academy of SciencesSt. PetersburgRussia

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