Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Summary

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Abstract

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.

Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

Introduction

LDs are bioactive organelles found in the cytosol of most eukaryotic cells and some prokaryotic cells^1,2,3. The LD core is composed of neutral lipids such as triglyceride (TG) and cholesterol esters. They also contain ceramides, bioactive lipids involved in cellular signaling pathways^4,5. LDs are surrounded by a phospholipid monolayer and coated with proteins, including the perilipin proteins perilipin 2 (PLIN2) and 3 (PLIN3)^1,5,6<....

Protocol

All experiments were conducted on a laboratory bench with personal protective equipment appropriate for biosafety level 1, including a lab coat, gloves, and safety goggles. Experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania. All efforts were made to minimize animal discomfort, and the animals were treated with humane care.

1. LD isolation using an ER isolation kit

1. Preparation
   NOTE: The listed amounts are suitable for a single 1 g mouse liver.
   1. Prepare 10 mL of 1x isotonic extraction buffer ....

Results

We have performed the LD isolation with the ER kit on approximately 30 mice and compared the results with those following the sucrose isolation protocol on approximately 40 mice. The reported findings are typical for both protocols. Mice were fasted overnight with free access to water, to increase the LD yield. LD isolation using a sucrose gradient was run in parallel with the ER kit LD isolation protocol. Samples of PNS, PMS, PER, CLDs, and LDs were collected throughout the ER kit LD iso.......

Discussion

Hepatic LD biology is increasingly being recognized as a critical regulator of hepatocellular physiology in both health and disease. As bona fide organelles, LDs are dynamic, interact with other cellular structures, and contain within them bioactive components involved in both lipid and glucose homeostasis. The sucrose gradient is routinely used for LD isolation and enables investigators to study LD structure, but it requires them to make numerous buffers. We have demonstrated that the use of a commercially available ER .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BioExpress Vortex Mixer	GeneMate	S-3200-1	Vortex Mixer
Centrifuge 54242 R	Eppendorf	54242 R	Refrigerated Counter Microcentrifuge Rotor: FA-45-24-11
Centrifuge Sorvall RC6 +	Thermo Scientific	RC6+	Refrigerated High Speed Centrifuge Rotor: F21S-8x50y
Centrifuge Sorvall Legend RT+	Thermo Scientific	Legend RT+	Refrigerated High Capacity Centrifuge Rotor: 75006445 Bucket: 75006441
cOmplete Protease Inhibitor Cocktail	Sigma Aldrich	04 693 116 001	Protease Inhibitor Tablets
Diagenode Bioruptur UCD-200	Diagenode	UCD-200	Sonication System
Dounce Tissue Grinder (45 mL)	Wheaton	357546	Use Loose pestle
Dulbecco's Phosphate-Buffered Saline (1x)	Corning	21-031-CM
Endoplasmic Reticulum Isolation Kit	Sigma Aldrich	ER0100	For ER kit Extraction: Contains: Isotonic Extraction Buffer 5X 100 mL Hypotonic Extraction Buffer 10 mL Calcium chloride, 2.5 M solution 5 mL OptiPrep Density Gradient Medium 100 mL Blunt Nosed Needle 1 ea.
External light source for flourescent excitation	Leica	Leica EL6000
Hydrochloric acid	Sigma Aldrich	H1758	Hydrochloric Acid
ImageJ			Image Analysis Software
Inverted Modulating Contrast Microscope	Leica	Leica DM IRB
Lysosome Enrichment Kit for Tissues and Cultured Cells	Thermo Fisher Scientific	89839	For Lysosome Isolation
Microscope Camera	Qimaging	QICAM fast 1394
Optima XL-100K Ultracentrifuge	Beckman-Coulter	XL-100K	Ultracentrifuge Rotor: SW41Ti
Pasteur Pipettes	Fisher Scientific	22-042817
Petri Dish 5 cm	Fisher Scientific	FB0875713A
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	23225
PhosSTOP	Sigma Aldrich	04 906 845 001	Phosphatase Inhibior Tablets
Sterile Surgical Blade #10	Pioneer	S2646
Sucrose	Fisher Scientific	S5-500	Crystalline/Certified ACS
Triglyceride Liquicolor Kit	Stanbio	2200-225	Colorimetric Triglyceride kit
Tris Base	Fisher Scientific	BP152-1	For TE buffer
Triton X-100	Fisher BioReagents	BP151-100	5%, Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, Protein Lysis Reagent
UltraPure EDTA (0.5M, pH 8.0)	Thermo Fisher Scientific	15575-038	For TE buffer