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 ...

Research into lipid droplet biology has been hampered by difficulty of purification. Additionally, studying cellular lipid flux requires simultaneous purification of multiple organelles, and no simple protocols were currently available. The main advantage of this technique is to make lipid droplet purification more accessible.We modified a commercially available kit to simultaneously isolate lipid droplets, endoplasmic reticulum, and lysosomes from a single sample. Liver lipid droplet accumulation predisposes obese and alcoholic patients to progressive liver disease. This has shifted the view of lipid droplets from inert storage compartments to dynamic biologically important organelles.To begin, use sterile forceps and dissection scissors to cut the skin and muscle layers of the abdomen of the euthanized mouse on the posterior-anterior axis, exposing the liver. Cut the ligaments connecting the liver to the intestine. Using forceps, pull the intestine away from the liver, toward the posterior.Cut the ligament connecting the liver to the diaphragm. Then, cut between the liver and the dorsal ribcage, moving from anterior to posterior, until the liver is liberated. Transfer the liver to a cold five-centimeter Petri dish with 10 milliliters of cold 1x PBS, and wash the liver twice on a rocking platform in 10 milliliters of cold 1x PBS for five minutes at four degrees Celsius.After the final wash, pour off the 1x PBS, and use a size-10 sterile surgical blade to dice the liver into three-to five-millimeter pieces within the dish. Then, wash the diced liver two times with 10 milliliters of cold 1x PBS for five minutes at four degrees Celsius. Decant the 1x PBS, and transfer the diced liver pieces to a cold 45-milliliter Dounce homogenizer, ensuring all pieces are at the bottom.Add seven milliliters of cold 1x IE buffer. Homogenize on ice by moving the pestle up and down 20 times, making sure that the pestle goes to the bottom of the homogenizer during each stroke. Transfer the homogenate to a cold 15-milliliter centrifuge tube.Rinse the homogenizer with one milliliter of cold 1x IE buffer, and add it to the rest of the homogenate. To remove nuclei, centrifuge the homogenate in a high-capacity centrifuge at 1, 000 times g for 10 minutes at four degrees Celsius. Ensure that any lipid gathered at the top of the 15-milliliter tube is resuspended to prevent LD loss.Making sure that nuclear pellet at the bottom of the tube is undisturbed, transfer the lipid-containing post-nuclear supernatant to a fresh cold 50-milliliter centrifuge tube. Transfer a 100-microliter aliquot of the post-nuclear supernatant to a cold 1.7-milliliter microcentrifuge tube on ice for later purity analysis. To remove mitochondria, centrifuge the post-nuclear supernatant sample in a refrigerated high-speed centrifuge at 12, 000 times g for 15 minutes at four degrees Celsius.Ensure that any lipid gathered at the top of the tube is resuspended to prevent LD loss, and then transfer the post-mitochondrial supernatant to a 13-milliliter ultracentrifuge tube. Transfer a 100-microliter aliquot of the post-mitochondrial supernatant to a cold 1.7-milliliter microcentrifuge tube on ice for later purity analysis. Fill the ultracentrifuge tube with post-mitochondrial supernatant to the brim with cold 1x IE buffer, to prevent it collapsing during ultracentrifugation.Balance the samples, and then centrifuge in an ultracentrifuge at 100, 000 times g for 60 minutes at four degrees Celsius. To remove the LD layer, tilt the tube at a 45-degree angle, and aspirate with a glass pipette. After transferring the pellet to a cold 1.7-milliliter microcentrifuge tube, collect 100 microliters of the post-ER supernatant under the lipid layer for purity analysis.To pellet any cell debris, centrifuge in a microcentrifuge at top speed for five minutes at four degrees Celsius. Then, warm the tube gently with hands to help resuspend the LD layer, and transfer the supernatant, including the lipid layer, to a new 1.7-milliliter microcentrifuge tube. Repeat these washing steps, warming the tube two to three times, until the pellet is no longer visible.Then, to wash the pellet-free LD supernatant, add 1x PBS to a final volume of 1.5 milliliters, and centrifuge in a microcentrifuge at top speed for five minutes at four degrees Celsius. Use a glass pipette to remove one milliliter of 1x PBS, which is underneath the lipid layer, without disturbing the lipid layer. Repeat LD washing four to five times, until the 1x PBS is visually transparent with no turbidity.Then, use a glass pipette to remove all 1x PBS below the lipid layer, and use the resulting pure LD fraction for downstream analysis. When representative 20x fluorescent and brightfield LD images were taken from both the ER kit LD isolation and the sucrose LD isolation methods, they revealed similar levels of particulate matter, suggesting similar purities using two different protocols. Following purification, LD triglyceride and protein levels were measured using colorimetric assays, and the data were normalized to liver weight, showing that when the starting material was normalized, LD triglyceride and protein yields were similar using the ER kit and the sucrose method.LD diameters were quantified using image analysis software, showing that the ER kit LD isolation and sucrose LD isolation yielded LDs of similar sizes. To assess LD purity, samples were assayed by immunoblotting, showing that the LDs derived from the ER kit LD isolation and the sucrose gradient protocol both had equal purity. PLIN2, an LD marker, was detected in all samples except for the ER kit LD isolation PER and the sucrose's PNS.Purity analysis of an ER fraction using immunoblots shows that they were free of LD marker PLIN2 but had significant levels of the ER SEC61A protein. Purity of a lysosomal fraction after further purification of lysosomes using the lysosome enrichment kit was also assessed. Discovery is currently the most useful application of our protocol.We perform lipidomics in purified organelles, and showed lipotoxins are increased specifically in lipid droplets in alcoholic liver disease.

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JoVE Journal

Biology

10.9K vistas.  Perelman School of Medicine, University of Pennsylvania. La investigación sobre la biología de las gotas lipídicas se ha visto obstaculizada por la dificultad de purificación. Además, el estudio del flujo de lípidos celulares requiere la purificación simultánea de múltiples orgánulos, y actualmente no había protocolos simples disponibles. La principal ventaja de esta técnica es hacer más accesible la purificación de gotas lipídicas.Modificamos un kit disponible comercialmente para aislar simultáneamente gotas de lípidos, ret...