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Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation

Dawn L. Brasaemle1,  Nathan E. Wolins2

1Rutgers, The State University of New Jersey, New Brunswick, New Jersey
2Washington University of St. Louis School of Medicine, St. Louis, Missouri


Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
UNIT 3.15
DOI: 
10.1002/0471143030.cb0315s29
Online Posting Date: 
January, 2006
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Abstract

Lipid droplets are organelles found in most mammalian cells, as well as various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol into which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation.

Keywords: Cell cultures; mammalian; preparation of lipid droplets from centrifugation; density-gradient centrifugation; isolation of lipid droplets

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Isolation of Lipid Droplets from Cultured Cells by Density Gradient Centrifugation
  • Alternate Protocol: Isolation of Lipid Droplets with Lysis of Cells Using a Cell Disruption Bomb
  • Support Protocol 1: Lipid Loading of Cultured Cells
  • Support Protocol 2: Solubilization of Lipid Droplet–Associated Proteins for Immunoblotting
  • Support Protocol 3: Delipidation and Solubilization of Lipid Droplet–Associated Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
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Materials

Basic Protocol: Isolation of Lipid Droplets from Cultured Cells by Density Gradient Centrifugation

 Materials
  • 4 to 10 100-mm dishes containing confluent monolayer cells (1–2.5 × 107 cells)
  • Phosphate-buffered saline (PBS; appendix 2A), ice cold
  • Hypotonic lysis medium (HLM; see recipe), ice cold
  • HLM containing 60% and 5% (w/w) sucrose (see recipes), ice cold
  • Rubber policeman or cell-scraper
  • 15-ml plastic tubes with caps
  • Potter-Elvehjem tissue homogenizer with loose-fitting Teflon pestle (Wheaton), 4-ml capacity, 0.01 to 0.02 cm clearance
  • Low-speed refrigerated centrifuge with swinging-bucket rotor and appropriate centrifuge tubes
  • Beckman or Sorvall ultracentrifuge with SW41Ti or Th-641 swinging-bucket rotor
  • 13.2-ml thin-walled polyallomer or polycarbonate ultracentrifuge tubes
  • Beckman tube slicer with two metal shim rings and two rubber rings to fit ultracentrifuge tubes
  • Additional reagents and equipment for SDS-PAGE (unit 6.1) and immunoblotting (unit 6.2)

Alternate Protocol: Isolation of Lipid Droplets with Lysis of Cells Using a Cell Disruption Bomb

 Additional Materials (also see Basic Protocol)
  • Suspended cells in HLM (see Basic Protocol, step )
  • 45-ml cell disruption bomb (Parr)
  • 15- and 50-ml plastic tubes

Support Protocol 1: Lipid Loading of Cultured Cells

 Materials
  • Fatty acid–free bovine serum albumin
  • 0.1 M Tris×Cl, pH 8.0
  • Oleic acid
  • 50-ml screw-capped polypropylene tubes
  • Rotisserie shaker
  • 0.2- or 0.45-µm filter unit

Support Protocol 2: Solubilization of Lipid Droplet–Associated Proteins for Immunoblotting

 Materials
  • 10% (w/v) sodium sodecyl sulfate (SDS; see recipe)
  • Fresh lipid droplet fraction (see Basic Protocol)
  • 2× SDS sample buffer (for discontinuous systems; see unit 6.1)
  • Sonicating water bath with adjustable temperature
  • Vortex mixer
  • 1.5-ml microcentrifuge tubes
  • Gel-loading pipet tip or wide-guage needle attached to a small syringe
  • Additional reagents and equipment for a discontinuous SDS-PAGE gel (see unit 6.1)

Support Protocol 3: Delipidation and Solubilization of Lipid Droplet–Associated Proteins

 Materials
  • Acetone, –80°C and room temperature
  • Frozen lipid droplet fraction, thawed
  • 1:1 (v/v) acetone/ether
  • Ether
  • 2× SDS sample buffer (for discontinuous systems; see unit 6.1)
  • Extra reducing reagent (e.g., -mercaptoethanol or dithiothreitol)
  • Polypropylene screw-capped centrifuge tubes (Sarstedt)
  • High-speed refrigerated centrifuge with Sorvall SS34 rotor and tube adapter sleeves, or equivalent
  • Sonicating water bath with adjustable temperature
  • 1.5-ml microcentrifuge tubes

NOTE:All procedures using organic solvents should be carried out in a fume hood. Tubes should be tightly capped before removing samples from the fume hood for incubation or centrifugation steps. Glass pipets and storage containers should be used to transfer solvents, because disposable polystyrene laboratory pipets will dissolve in many organic solvents. Solvent waste should be disposed of in accordance with institutional policy.

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Figures

  • Figure 3.15.1
    Cultured 3T3-L1 adipocyte labeled with fluorescent fatty acids (A and C: green) and stained for perilipin (B and C: red). Cultured 3T3-L1 adipocytes were differentiated for 6 days with the addition of a BODIPY-labeled 12-carbon fatty acid (Molecular Probes, Invitrogen detection technologies, D3822, BODIPYFL C12) for the final 18 hr. Cells were fixed with 2% paraformaldehyde in PBS prior to staining with guinea pig polyclonal antibody to perilipin (Research Diagnostics, Inc., RDI-PROGP29) followed by AlexaFluor 594 goat anti-guinea pig IgG (Molecular Probes, Invitrogen detection technologies, A11076). Images were captured with a Zeiss Axioplan-2 microscope equipped with a Hamamatsu Orca CCD camera.

    View Image
  • Figure 3.15.2
    Coomassie Blue-stained proteins of lipid droplets isolated from 3T3-L1 adipocytes incubated under basal, lipid-storing conditions (A) and lipolytically stimulated conditions (B). Positions of molecular mass markers and stained bands are depicted on the left and right sides of the panels, respectively. Figure reprinted with permission of The Journal of Biological Chemistry, from “Proteomic Analysis of Proteins Associated with Lipid Droplets of Basal and Lipolytically Stimulated 3T3-L1 Adipocytes,” Dawn L. Brasaemle, Georgia Dolios, Lawrence Shapiro, and Rong Wang, Vol. 279 (2004) 46835-46842 (original manuscript); Correction Vol. 280 (2005) 4004; permission conveyed through Copyright Clearance Center, Inc.

    View Image

Literature Cited

Literature Cited
    Bligh, E.G. and Dyer, W.J. 1959. A rapid method of total lipid extraction and purification. Can. J. Med. Sci. 37:911-917.
    Brasaemle, D.L., Barber, T., Kimmel, A.R., and Londos, C. 1997a. Post-translational regulation of perilipin expression. Stabilization by stored intracellular neutral lipids. J. Biol. Chem. 272:9378-9387.
    Brasaemle, D.L., Barber, T., Wolins, N.E., Serrero, G., Blanchette-Mackie, E.J., and Londos, C. 1997b. Adipose differentiation-related protein is a ubiquitously expressed lipid storage droplet-associated protein. J. Lipid Res. 38:2249-2263.
    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.
    Caldas, H. and Herman, G.E. 2003. NSDHL, an enzyme involved in cholesterol biosynthesis, traffics through the Golgi and accumulates on ER membranes and on the surface of lipid droplets. Hum. Mol. Genet. 12:2981-2991.
    Coleman, R.A. and Lee, D.P. 2004. Enzymes of triacylglycerol synthesis and their regulation. Prog. Lipid Res. 43:134-176.
    Fujiki, Y., Hubbard, A.L., Fowler, S., and Lazarow, P.B. 1982. Isolation of intracellular membranes by means of sodium carbonate treatment: Application to endoplasmic reticulum. J. Cell Biol. 93:97-102.
    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.
    Gocze, P.M. and Freeman, D.A. 1994. Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells. Cytometry 17:151-158.
    Greenspan, P., Mayer, E.P., and Fowler, S.D. 1985. Nile red: A selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100:965-973.
    Liu, P., Ying, Y., Zhao, Y., Mundy, D.I., Zhu, M., and Anderson, R.G. 2004. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 279:3787-3792.
    Ohashi, M., Mizushima, N., Kabeya, Y., and Yoshimori, T. 2003. Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets. J. Biol. Chem. 278:36819-36829.
    Petitpas, I., Grune, T., Bhattacharya, A.A., and Curry, S. 2001. Crystal structures of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids. J. Mol. Biol. 314:955-960.
    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.
     
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