Name: rapid lipid droplet isolation protocol using a well-established organelle isolation kit
Uploaded: 2019-04-19T14:00:00
Description: Watch this Scientific Journal Video about Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit at JoVE.com

We thank the Abramson Family Cancer Research Institute. This work is supported by following grants: NIH/NIAAA R01 AA026302-01, NIH/NIAAA K08-AA021424, Robert Wood Johnson Foundation, Harold Amos Medical Faculty Development Award, 7158, IDOM DRC Pilot Award&#160;P30 DK019525 (R.M.C.); NIH/NIAAA F32-AA024347 (J.C.). This work was supported in part by NIH P30-DK050306 and its core facilities (Molecular Pathology and Imaging Core, Molecular Biology/Gene Expression Core, and Cell Culture Core) and its pilot grant program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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
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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 ...

LDs are bioactive organelles found in the cytosol of most eukaryotic cells and some prokaryotic cells1,2,3. 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 pathways4,5. LDs are surrounded by a phospholipid monolayer and coated with proteins, including the perilipin proteins perilipin 2 (PLIN2) and 3 (PLIN3)1,5,6. Also present are lipogenic enzymes, lipases, and membrane trafficking proteins that have been linked to several hepatic steatotic diseases, including nonalcoholic fatty liver disease, alcoholic liver disease, and hepatitis C3,5,6,7,8,9,10.
LDs are thought to form from the outer membrane of the ER and contain newly synthesized TG sourced from ER-derived free fatty acids11. However, it remains unknown whether the pooled lipids bud, remain connected, or are excised from the ER6,11, making the isolation of LDs free from ER technically challenging. Free fatty acids can be liberated from LDs by the action of surface lipases to provide for energy production or membrane synthesis. Additionally, LDs can be degraded via lipophagy as a regulatory mechanism and to produce free fatty acids12.
Besides the ER and lysosomes, there are other organelles&#8212;such as mitochondria, endosomes, peroxisomes, and the plasma membrane&#8212;that are found within close association of LDs11. This tight polygamy makes it difficult to perform a pure LD extraction. However, neutral lipids&#8217; innate low density can be taken advantage of via centrifugation5.
Traditionally, LDs have been isolated using a sucrose density gradient1,5,13. However, these methods have not been designed to isolate other organelles. Additionally, they require the time-consuming preparation of reagents. This protocol adapts a commercially available ER isolation kit (see the Table of Materials) to allow for LD isolation. We use the extraction buffer provided by the kit, adding an LD isolation step. Furthermore, the protocol can also be used for ER and lysosomal isolation using the same samples, allowing for a more comprehensive image of the life cycle of LDs. To validate this new method, we assay LD yield, morphology, size, and purity. We compare the results presented here to those obtained with a widely used protocol utilizing a sucrose gradient for LD isolation.
We used a 10- to 12-week-old, 20 g female C57BL/6 mouse fasted for 16 h (food removal at 5.00 P.M. for a 9.00 A.M. LD isolation time) with free access to water. The typical yield for TG is 0.6 mg/g liver, and for LD protein, it is 0.25 mg/g liver. This provides enough material for ~10 immunoblots by SDS PAGE. The protocol below details the reagents used and a protocol suitable for a single 1 g liver. The sucrose gradient isolation protocol is adapted from Sato14 and Wu15.

<table border="0" cellpadding="0" cellspacing="0" style="width:928px;" width="927">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">BioExpress Vortex Mixer</td>
			<td style="width:123px;">GeneMate</td>
			<td style="width:344px;">S-3200-1</td>
			<td style="width:245px;">Vortex Mixer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge 54242 R</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:344px;">54242 R</td>
			<td style="width:245px;">Refrigerated Counter Microcentrifuge 
			Rotor: FA-45-24-11</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge Sorvall RC6 +</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:344px;">RC6+</td>
			<td style="width:245px;">Refrigerated High Speed Centrifuge 
			Rotor: F21S-8x50y</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge Sorvall Legend RT+</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:344px;">Legend RT+</td>
			<td style="width:245px;">Refrigerated High Capacity Centrifuge 
			Rotor: 75006445 Bucket: 75006441</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">cOmplete Protease Inhibitor Cocktail</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:344px;">04 693 116 001</td>
			<td style="width:245px;">Protease Inhibitor Tablets</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Diagenode Bioruptur UCD-200</td>
			<td style="width:123px;">Diagenode</td>
			<td style="width:344px;">UCD-200</td>
			<td style="width:245px;">Sonication System</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dounce Tissue Grinder (45 mL)</td>
			<td style="width:123px;">Wheaton</td>
			<td style="width:344px;">357546</td>
			<td style="width:245px;">Use Loose pestle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dulbecco&#39;s Phosphate-Buffered Saline (1x)</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">21-031-CM</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:216px;">Endoplasmic Reticulum Isolation Kit</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:344px;">ER0100</td>
			<td style="width:245px;">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.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">External light source for flourescent excitation</td>
			<td style="width:123px;">Leica</td>
			<td style="width:344px;">Leica EL6000</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Hydrochloric acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:344px;">H1758</td>
			<td style="width:245px;">Hydrochloric Acid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">ImageJ</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:245px;">Image Analysis Software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Inverted Modulating Contrast Microscope</td>
			<td style="width:123px;">Leica</td>
			<td style="width:344px;">Leica DM IRB</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Lysosome Enrichment Kit for Tissues and Cultured Cells</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:344px;">89839</td>
			<td style="width:245px;">For Lysosome Isolation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Microscope Camera</td>
			<td style="width:123px;">Qimaging</td>
			<td style="width:344px;">QICAM fast 1394</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Optima XL-100K Ultracentrifuge</td>
			<td style="width:123px;">Beckman-Coulter</td>
			<td style="width:344px;">XL-100K</td>
			<td style="width:245px;">Ultracentrifuge 
			Rotor: SW41Ti</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Pasteur Pipettes</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">22-042817</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Petri Dish 5 cm</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">FB0875713A&#160;</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:344px;">23225</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PhosSTOP</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:344px;">04 906 845 001</td>
			<td style="width:245px;">Phosphatase Inhibior Tablets</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sterile Surgical Blade #10</td>
			<td style="width:123px;">Pioneer</td>
			<td style="width:344px;">S2646</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sucrose</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">S5-500</td>
			<td style="width:245px;">Crystalline/Certified ACS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Triglyceride Liquicolor Kit</td>
			<td style="width:123px;">Stanbio&#160;</td>
			<td style="width:344px;">2200-225</td>
			<td style="width:245px;">Colorimetric Triglyceride kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Tris Base</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">BP152-1</td>
			<td style="width:245px;">For TE buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Triton X-100</td>
			<td style="width:123px;">Fisher BioReagents</td>
			<td style="width:344px;">BP151-100</td>
			<td style="width:245px;">5%, Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, Protein Lysis Reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">UltraPure EDTA (0.5M, pH 8.0)</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:344px;">15575-038</td>
			<td style="width:245px;">For TE buffer</td>
		</tr>
	</tbody>
</table>

rapid lipid droplet isolation protocol using a well-established organelle isolation kit

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.

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...

Watch this Scientific Journal Video about Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit at JoVE.com

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

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

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

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