Fluorescent Detection of Lipid Droplets and Associated Proteins

Laura L. Listenberger1, Andrea M. Studer1, Deborah A. Brown2, Nathan E. Wolins3

1 Departments of Biology and Chemistry, St. Olaf College, Northfield, Minnesota, 2 Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, 3 Center for Human Nutrition, Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.31
DOI:  10.1002/cpcb.7
Online Posting Date:  June, 2016
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Abstract

Excess lipid is stored in intracellular organelles known as 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 in fixed or live cells with BODIPY 493/503 are included. 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 perilipin 2, 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. © 2016 by John Wiley & Sons, Inc.

Keywords: BODIPY 493/503; lipid droplets; lipid droplet proteins; perilipin 2

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Lipid Droplets with BODIPY 493/503
  • Basic Protocol 2: Simultaneous Detection of Lipid Droplets and Perilipin 2, a Lipid Droplet–Associated Protein
  • Support Protocol 1: Preparation of Fatty Acid–Supplemented Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of Lipid Droplets with BODIPY 493/503

  Materials
  • Cultured cells
  • Growth medium, optionally supplemented with 400 μM sodium oleate (see protocol 3Support Protocol)
  • 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 for 2 months at 4°C
  • 1 mg/ml BODIPY 493/503 stock solution: prepare by dissolving 10 mg BODIPY 493/503 (4,4‐difluoro‐1,3,5,7,8‐pentamethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene) in 10 ml ethanol; divide into 500‐μl aliquots, and store for 1 year at −20°C
  • 10 mg/ml Hoechst 33342 or 10 mg/ml DAPI (4′,6‐diamidino‐2‐phenylindole, dihydrochloride), dissolved in H 2O
  • Treatments of interest
  • 3% (w/v) paraformaldehyde (see recipe), or 2% formaldehyde prepared by adding 13.5 ml of 37% formaldehyde to 236.5 ml PBS
  • Phosphate‐buffered saline (PBS; appendix 2A), without calcium and magnesium
  • Microscopy buffer (see recipe)
  • Mounting medium: e.g., Elvanol (Sigma, cat. no. 10981; Wessel et al., ) or Prolong Gold Antifade Reagent, optionally with DAPI stain
  • Nail polish, clear and uncolored, such as a top coat (Sally Hansen or other; optional)
  • 22‐mm2 square glass coverslips (1.5 thickness), sterilized by baking at 160°C for at least 2 hr in aluminum foil covered glass beaker
  • 6‐well tissue culture plates or 35‐mm‐diameter tissue culture dishes
  • Confocal or wide‐field fluorescence microscope with appropriate filters
  • Glass slides
  • Additional reagents and equipment for preparing medium supplemented with oleate ( protocol 3Support Protocol)

Basic Protocol 2: Simultaneous Detection of Lipid Droplets and Perilipin 2, a Lipid Droplet–Associated Protein

  Materials
  • Cultured cells
  • Growth medium, optionally supplemented with 400 μM sodium oleate (see protocol 3Support Protocol)
  • 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 for 2 months at 4°C
  • Treatments of interest
  • 3% (w/v) paraformaldehyde (see recipe), or 2% formaldehyde prepared by adding 13.5 ml of 37% formaldehyde to 236.5 ml PBS
  • Phosphate‐buffered saline (PBS; appendix 2A), without calcium and magnesium
  • Microscopy buffer (see recipe)
  • Primary antibody: Anti‐perilipin‐2 (Santa Cruz Biotechnology, cat. no. sc‐377429)
  • 1 mg/ml BODIPY 493/503 stock solution: prepare by dissolving 10 mg BODIPY 493/503 (4,4‐difluoro‐1,3,5,7,8‐pentamethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene) in 10 ml ethanol; divide into 500‐μl aliquots, and store for 1 year at −20°C
  • Secondary antibody: Alexa Fluor 594 donkey anti‐mouse IgG (Jackson ImmunoResearch Laboratories, cat. no. 715‐585‐150)
  • 22‐mm2 square glass coverslips (1.5 thickness), sterilized by baking at 160°C for at least 2 hr in aluminum foil covered glass beaker
  • 6‐well tissue culture plates or 35‐mm‐diameter tissue culture dishes
  • Additional reagents and equipment for plating cells and mounting and imaging coverslip with cells ( protocol 1)

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 (Sigma, cat. no. A6003)
  • Growth medium for cultured cells of interest
  • 50‐ml conical tube (e.g., Corning Falcon)
  • 37°C and 70°C water bath
  • 250‐ and 400‐ml beakers, sterile
  • 0.22‐μm bottle‐top filter
  • 200‐ml glass bottle, sterile
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Figures

Videos

Literature Cited

Literature Cited
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