Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation

Dawn L. Brasaemle1, Nathan E. Wolins2

1 Rutgers, The State University of New Jersey, New Brunswick, 2 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.15
DOI:  10.1002/cpcb.10
Online Posting Date:  September, 2016
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Abstract

Lipid droplets are organelles found in most mammalian cells, as well as in various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol in which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation. © 2016 by John Wiley & Sons, Inc.

Keywords: cell cultures; mammalian; preparation of lipid droplets by centrifugation; density‐gradient centrifugation; isolation of lipid droplets

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Lipid Droplets from Cultured Cells by Density Gradient Centrifugation
  • Alternate Protocol 1: 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
     
 
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Materials

Basic Protocol 1: 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 reciperecies), 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; Gallagher, ) and immunoblotting (unit 6.2; Gallagher et al., )

Alternate Protocol 1: Isolation OF Lipid Droplets with Lysis of Cells using a cell Disruption Bomb

  Additional Materials (also see protocol 1Basic Protocol)
  • Suspended cells in HLM (see protocol 1Basic Protocol, step 4)
  • 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 ( appendix 2A)
  • 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 dodecyl sulfate (SDS; see recipe)
  • Fresh lipid droplet fraction (see protocol 1Basic Protocol)
  • 2× SDS sample buffer [for discontinuous systems; see unit 6.1 (Gallagher, )]
  • Sonicating water bath with adjustable temperature
  • Vortex mixer
  • 1.5‐ml microcentrifuge tubes
  • Gel‐loading pipet tip or wide‐gauge needle attached to a small syringe
  • Additional reagents and equipment for a discontinuous SDS‐PAGE gel (see unit 6.1; Gallagher, )

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; Gallagher, ]
  • Extra reducing reagent (e.g., 2‐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

Videos

Literature Cited

Literature Cited
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