Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation

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; density-gradient centrifugation; isolation of lipid droplets; mammalian; preparation of lipid droplets by centrifugation.

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®, ThermoFisher Scientific, D-3822, BODIPY® FL C₁₂) for the final 18 hr. Cells were fixed with 2% paraformaldehyde in PBS prior to staining with guinea pig polyclonal antibody to perilipin (Fitzgerald Industries International, 20R-PP004 or Progen Biotechnik, GP29) followed by AlexaFluor 594 goat anti-guinea pig IgG (ThermoFisher Scientific, A-11076). Images were captured with a Zeiss Axioplan-2 microscope equipped with a Hamamatsu Orca CCD camera. For the color version of this figure go to http://www.currentprotocols.com.

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.