Fluorescent Detection of Lipid Droplets and Associated Proteins

Laura L. Listenberger1, Deborah A. Brown1

1 State University of New York at Stony Brook, Stony Brook, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 24.2
DOI:  10.1002/0471143030.cb2402s35
Online Posting Date:  June, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Most eukaryotic cells can store excess lipid in cytosolic lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets with nile red and BODIPY 493/503 are included. The differences in the spectral properties of these two lipophilic dyes and advantages of each are discussed. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet–associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of adipophilin, a broadly expressed, lipid droplet–associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included.

Keywords: lipid droplets; nile red; BODIPY 493/503; adipophilin; PAT proteins

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Detection of Lipid Droplets with Lipophilic Dyes
  • Basic Protocol 2: Immunofluorescent Detection of Adipophilin, a Lipid Droplet–Associated Protein
  • Alternate Protocol 1: Simultaneous Detection of Lipid Droplets and Adipophilin
  • Support Protocol 1: Preparation of Fatty Acid–Supplemented Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Detection of Lipid Droplets with Lipophilic Dyes

  Materials
  • Cultured cells
  • Poly‐L‐lysine, optional: prepare by dissolving 5 mg poly‐L‐lysine in 50 ml sterile H 2O; sterilize by passing through a 0.22‐µm filter, and store up to 2 months at 4°C
  • Growth medium, optionally supplemented with 400 µM oleate (see protocol 4)
  • Phosphate‐buffered saline (PBS; appendix 2A), without calcium and magnesium
  • 3% (w/v) paraformaldehyde in PBS
  • 1 mg/ml BODIPY 493/503 stock solution: prepare by dissolving 5 mg BODIPY 493/503 (4,4‐difluoro‐1,3,5,7,8‐pentamethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene; Molecular Probes) in 5 ml ethanol; divide into 500‐µl aliquots, and store up to 1 month at −20°C or
  • 1 mg/ml Nile Red stock solution: prepare by dissolving 5 mg Nile Red (Molecular Probes) in 5 ml DMSO; divide into 500‐µl aliquots, and store up to 1 month at −20°C
  • 150 mM NaCl: prepare by dissolving 4.38 g NaCl in 500 ml H 2O; store up to 1 year at room temperature
  • Mounting medium: e.g., Prolong Antifade Reagent (Invitrogen) or see
  • 22‐mm2 square glass coverslips, sterile
  • 35‐mm tissue culture dishes or 6‐well tissue culture plates
  • Glass slides
  • Fluorescence microscope with appropriate filters

Basic Protocol 2: Immunofluorescent Detection of Adipophilin, a Lipid Droplet–Associated Protein

  Materials
  • Cultured cells
  • Growth medium, optionally supplemented with 400 µM oleate (see protocol 4)
  • 0.1 mg/ml poly‐L‐lysine, optional: prepare by dissolving 5 mg poly‐L‐lysine in 50 ml sterile H 2O; sterilize by passing through a 0.22‐µm filter, and store up to 1 month at 4°C
  • Phosphate‐buffered saline (PBS; appendix 2A), without calcium and magnesium
  • 3% (w/v) paraformaldehyde in PBS
  • Blocking buffer: 0.2 M glycine /0.1 mg/ml saponin/30 mg/ml bovine serum albumin (BSA) in PBS ( appendix 2A)
  • Primary antibody: guinea pig anti‐adipophilin polyclonal antibody (Research Diagnostics)
  • Antibody diluent: 0.1 mg/ml saponin /1 mg/ml BSA in PBS ( appendix 2A)
  • Secondary antibody: fluorescently‐tagged anti‐guinea pig IgG; e.g., Texas red dye–conjugated donkey anti‐guinea pig IgG (Jackson Immunoresearch Laboratories)
  • Mounting medium: e.g., Prolong Antifade Reagent (Invitrogen) or see
  • 22‐mm2 square glass coverslips, sterile
  • 35‐mm tissue culture dishes or 6‐well tissue culture plates
  • Rocking platform
  • Glass slides
  • Confocal or widescreen fluorescence microscope with appropriate filters

Alternate Protocol 1: Simultaneous Detection of Lipid Droplets and Adipophilin

  • 1 mg/ml BODIPY 493/503 stock solution: prepare by dissolving 5 mg BODIPY 493/503 (4,4‐difluoro‐1,3,5,7,8‐pentamethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene; Molecular Probes) in 5 ml ethanol; divide into 500‐µl aliquots, and store up to 1 month at −20°C

Support Protocol 1: Preparation of Fatty Acid–Supplemented Medium

  Materials
  • 1 M NaOH
  • Oleic acid
  • Phosphate‐buffered saline (PBS; appendix 2A), without calcium and magnesium
  • Bovine serum albumin (BSA), essentially fatty acid free
  • Growth medium for cultured cells of interest
  • 50‐ml conical tube
  • 37°C and 70°C water bath
  • 250‐ and 400‐ml beakers, sterile
  • 0.22‐µm bottle‐top filter and sterile, 200‐ml glass bottle
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Aoki, T., Hagiwara, H., and Fujimoto, T. 1997. Peculiar distribution of fodrin in fat‐storing cells. Exp. Cell Res. 234:313‐320.
   Brasaemle, D.L., Dolios, G., Shapiro, L., and Wang, R. 2004. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3‐L1 adipocytes. J. Biol. Chem. 279:46835‐46842.
   Deye, J.F., Berger, T.A., and Anderson, A.G. 1990. Nile Red as a solvatochromic dye for measuring solvent strength in normal liquids and mixtures of normal liquids with supercritical and near critical fluids. Anal. Chem. 62:615‐622.
   DiDonato, D. and Brasaemle, D.L. 2003. Fixation methods for the study of lipid droplets by immunofluorescence microscopy. J. Histochem. Cytochem. 51:773‐780.
   Fukumoto, S. and Fujimoto, T. 2002. Deformation of lipid droplets in fixed samples. Histochem. Cell Biol. 118:423‐428.
   Fujimoto, Y., Itabe, H., Sakai, J., Makita, M., Noda, J., Mori, M., Higashi, Y., Kojima, S., and Takano, T. 2004. Identification of major proteins in the lipid droplet–enriched fraction isolated from the human hepatocyte cell line HuH7. Biochim. Biophys. Acta. 1644:47‐59.
   Gao, J., Ye, H., and Serrero, G. 2000. Stimulation of adipose differentiation related protein (ADRP) expression in adipocyte precursors by long‐chain fatty acids. J. Cell Phys. 182:297‐302.
   Greenspan, P., Mayer, E.U., and Fowler, S.D. 1985. Nile Red: A selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100:965‐973.
   Imamura, M., Inoguchi, T., Ikuyama, S., Taniguchi, S., Kobayashi, K., Nakashima, N., and Nawata, H. 2002. ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts. Am. J. Physiol. Endocrinol. Metab. 283:E775‐E783.
   Kleinfeld, A.M., Prothro, D., Brown, D.L., Davis, R.C., Richieri, G.V., and DeMaria, A. 1996. Increases in serum unbound free fatty acid levels following coronary angioplasty. Am. J. Cardiol. 78:1350‐1354.
   Koopman, R., Schaart, G., and Hesselink, M.K.C. 2001. Optimisation of oil‐red O staining permits combination with immunofluorescence and automated quantification of lipids. Histochem. Cell Biol. 116:63‐68.
   Larigauderie, G., Furman, C., Jaye, M., Lasselin, C., Copin, C., Fruchart, J., Castro, G., and Rouis, M. 2004. Adipophilin enhances lipid accumulation and prevents lipid efflux from THP‐1 macrophages: Potential role in atherogenesis. Arterioscler. Thromb. Vasc. Biol. 24:504‐510.
   Listenberger, L.L., Ory, D.S., and Schaffer, J.E. 2001. Palmitate‐induced apoptosis can occur through a ceramide‐independent pathway. J. Biol. Chem. 276:14890‐14895.
   Liu, P., Ying, Y., Zhao, Y., Mundy, D., Zhu, M., and Anderson, R.G.W. 2004. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 279:3787‐3792.
   Londos, C., Brasaemle, D.L., Schultz, C.J., Segrest, J.P., and Kimmel, A.R. 1999. Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells. Semin. Cell Dev. Biol. 10:51‐58.
   Londos, C., Sztalryd, C., Tansey, J.T., and Kimmel, A.R. 2005. Role of PAT proteins in lipid metabolism. Biochimie 87:45‐49.
   Murphy, D.J. and Vance, J. 1999. Mechanisms of lipid‐body formation. Trends Biol. Sci. 24:109‐115.
   Ohsaki, Y., Maeda, T., and Fujimoto, T. 2005. Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins. Histochem. Cell Biol. 124:445‐452.
   Ostermeyer, A.G., Paci, J.M., Zeng, Y., Lublin, D.M., Munro, S., and Brown, D.A. 2001. Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets. J. Cell Biol. 152:1071‐1078.
   Ostermeyer, A.G., Ramcharan, L.T., Zeng, Y., Lublin, D.M., and Brown, D.A. 2004. Role of the hydrophobic domain in targeting caveolin‐1 to lipid droplets. J. Cell Biol. 164:69‐78.
   Richieri, G.V. and Kleinfeld, A.M. 1995. Unbound free fatty acid levels in human serum. J. Lipid Res. 36:229‐240.
   Spector, A.A. 1975. Fatty acid binding to plasma albumin. J. Lipid Res. 16:165‐179.
   Targett‐Adams, P., Chambers, D., Gledhill, S., Hope, R.G., Coy, J.F., Girod, A., and McLauchlan, J. 2003. Live cell analysis and targeting of the lipid droplet–binding adipocyte differentiation‐related protein. J. Biol. Chem. 278:15998‐16007.
   Umlauf, E., Csaszar, E., Moertelmaier, M., Schuetz, G.J., Parton, R.G., and Prohaska, R. 2004. Association of stomatin with lipid bodies. J. Biol. Chem. 279:23699‐23709.
   Wolins, N.E., Quaynor, B.K., Skinner, J.R., Schoenfish, M.J., Tzekov, A., and Bickel, P.E. 2005. S3‐12, adipophilin, and TIP47 package lipid in adipocytes. J. Biol. Chem. 280:19146‐19155.
   Xu, G., Sztalryd, C., Lu, X., Tansey, J.T., Gan, J., Dorward, H., Kimmel, A.R., and Londos, C. 2005. Post‐translational regulation of ADRP by the ubiquitin/proteasome pathway. J. Biol. Chem. 280:42841‐42847.
   Zweytick, D., Athenstaedt, K., and Daum, G. 2000. Intracellular lipid particles of eukaryotic cells. Biochim. Biophys. Acta. 1469:101‐120.
Internet Resources
  http://spectorlab.cshl.edu/fluorescence_medium.html
  Includes detailed instructions for preparation of a p‐phenylenediamine containing fluorescence mounting medium.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library