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 ...

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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Biology

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from&#160;lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.