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
<ol>
	<li>Preparation 
	NOTE: The listed amounts are suitable for a single 1 g mouse liver.
	<ol>
		<li>Prepare 10 mL of 1x isotonic extraction buffer (IE buffer, from the ER isolation kit) by diluting 2 mL of 5x IE buffer with 8 mL of ultrapure water. Cool the buffer by placing it on ice.</li>
		<li>Prepare 100 mL of 1x phosphate-buffered saline (PBS) by diluting 10 mL of 10x PBS in 90 mL of ultrapure water. Cool it by placing it on ice.</li>
		<li>Prepare 10 mL of 5% polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (PGPE) by diluting 500 &#181;L of PGPE in 9.5 mL of ultrapure water. Use a 1 mL syringe to measure out PGPE as it is viscous. Cool it by placing it on ice.</li>
		<li>Cool all tubes by placing them on ice: 1.7 mL microcentrifuge tubes, 15 mL centrifuge tubes, 50 mL high-speed centrifuge tubes, and 13 mL ultracentrifuge tubes.</li>
		<li>Cool all required centrifuges to 4 &#176;C: a microcentrifuge, a high-capacity centrifuge for 15 mL tubes, a high-speed centrifuge for 50 mL tubes, and an ultracentrifuge.</li>
		<li>Sterilize forceps and dissection scissors by autoclaving.</li>
	</ol>
	</li>
	<li>Tissue preparation 
	<ol>
		<li>Euthanize the mouse by placing it in a chamber filled with 100% CO&#173;2 for 10 min. Confirm euthanasia by performing cervical dislocation.</li>
		<li>Using sterile forceps and dissection scissors, cut the skin and muscle layers of the abdomen on the posterior/anterior axis to expose the liver.</li>
		<li>Cut the ligaments connecting the liver to the diaphragm and intestine. Use forceps to pull the intestine away from the liver, toward the posterior, to expose the ligaments.</li>
		<li>Cut between the liver and the dorsal ribcage, moving from anterior to posterior until the liver is liberated.</li>
		<li>Transfer the liver to a cold 5 cm Petri dish with 10 mL of cold 1x PBS.</li>
		<li>Wash the liver 2x on a rocking platform in 10 mL of cold 1x PBS for 5 min at 4 &#176;C in the 5 cm Petri dish. Pour off 1x PBS after the final wash.</li>
		<li>Use a size-10 sterile surgical blade (see the Table of Materials) to dice the liver into 3&#8211;5 mm pieces in the 5 cm Petri dish (Figure 1A).</li>
		<li>Wash the diced liver 2x with 10 mL of cold 1x PBS for 5 min at 4 &#176;C in the 5 cm Petri dish. 
		NOTE: For lysosome isolation, transfer 0.5 g of liver to a clean 45 mL homogenizer and follow the directions supplied by the lysosome extraction kit (see the Table of Materials).</li>
		<li>Decant 1x PBS and transfer the diced liver pieces to the cold 45 mL Dounce homogenizer. Ensure all pieces go to the bottom of the homogenizer.</li>
		<li>Add 7 mL of cold 1x IE buffer (from the ER isolation kit) and homogenize on ice by moving the pestle up and down 20x. Ensure that the pestle goes to the bottom of the homogenizer during each stroke.</li>
		<li>Transfer the homogenate (Figure 1B) to a cold 15 mL centrifuge tube.</li>
		<li>Rinse the homogenizer with 1 mL of cold 1x IE buffer (from the ER isolation kit) and add this to the rest of the homogenate.</li>
	</ol>
	</li>
	<li>LD isolation 
	<ol>
		<li>Remove nuclei by centrifuging the homogenate in a high-capacity centrifuge at 1,000 x g for 10 min at 4 &#176;C (Figure 1C).</li>
		<li>Transfer the postnuclear supernatant (PNS) to a new, cold 50 mL centrifuge tube. To prevent LD loss, ensure that any lipid gathered at the top of the 15 mL tube is resuspended before transferring the PNS.</li>
		<li>Take a 100 &#181;L aliquot of the PNS for purity analysis and place it in a cold 1.7 mL microcentrifuge tube. Set it aside on ice.</li>
		<li>To remove mitochondria, centrifuge the PNS sample, not the aliquot, in a refrigerated high-speed centrifuge at 12,000 x g for 15 min at 4 &#176;C.
		<ol>
			<li>Optional: For crude LD (CLD) isolates (with cytosol and cell membrane contamination), collect the top lipid layer using a glass pipette and transfer it to a 1.7 mL microcentrifuge tube. Continue to section 1.4.</li>
		</ol>
		</li>
		<li>Transfer the postmitochondrial supernatant (PMS) to an ultracentrifuge tube. To prevent LD loss, ensure that any lipid gathered at the top is resuspended before transferring the PMS.</li>
		<li>Take a 100 &#181;L aliquot of the PMS for purity analysis and place it in a cold 1.7 mL microcentrifuge tube. Set it aside on ice.</li>
		<li>Fill an ultracentrifuge tube to the brim with cold 1x IE buffer to prevent the tube collapsing during ultracentrifugation.</li>
		<li>Balance the samples and centrifuge them in an ultracentrifuge at 100,000 x g for 60 min at 4 &#176;C (Figure 1D).</li>
		<li>To remove the LD layer, tilt the tube at a 45&#176; angle and use a glass pipette to aspirate. Transfer the pellet to a cold 1.7 mL microcentrifuge tube. 
		NOTE: The pellet contains isolated ER. Use this fraction downstream for ER isolation.</li>
		<li>Collect 100 &#181;L of the post-ER supernatant (PER) under the lipid layer for purity analysis.</li>
	</ol>
	</li>
	<li>LD washing
	<ol>
		<li>Add 1x PBS to the LD fraction collected in a 1.7 microcentrifuge tube, to a total volume of 1.3 mL.</li>
		<li>Vortex the sample.</li>
		<li>Centrifuge in a microcentrifuge at top speed for 5 min at 4 &#176;C to pellet any cell debris. Warm the tube gently with hands to help resuspend the LD layer. Transfer the supernatant, including the lipid layer, to a new 1.7 mL microcentrifuge tube.</li>
		<li>Repeat steps 1.4.1&#8211;1.4.3 2x&#8211;3x, until the pellet is no longer visible.</li>
		<li>Add 1x PBS to the pellet-free LD supernatant to a final volume of 1.5 mL. Wash the lipid fraction by centrifugation in a microcentrifuge at top speed for 5 min at 4 &#176;C.</li>
		<li>Remove 1 mL of 1x PBS (underneath the lipid layer) with a glass pipette without disturbing the lipid layer.</li>
		<li>Repeat steps 1.4.5 and 1.4.6 4x&#8211;5x, until the 1x PBS is visually transparent with no turbidity.</li>
		<li>Remove all 1x PBS below the lipid layer, using a glass pipette (Figure 1E).</li>
		<li>Use the result, a pure LD fraction, for downstream analysis.</li>
	</ol>
	</li>
	<li>LD protein quantitation by bicinchoninic acid assay 
	NOTE: Do not use a bicinchoninic acid assay (BCA) if LD morphology is to be assessed.
	<ol>
		<li>Resuspend the LD in 500 &#956;L of 5% PGPE.</li>
		<li>Boil the samples for 5 min at 95 &#176;C, vortex them, and incubate the samples at room temperature for 5 min to cool.</li>
		<li>Repeat step 1.5.2 an additional 2x.</li>
		<li>Sonicate the samples for 5 min with a 30 s on/off cycle, at medium intensity.</li>
		<li>Normalize the samples by measuring the protein concentration in all purity analysis samples and in the LD fraction by BCA assay.</li>
	</ol>
	</li>
</ol>
2. LD isolation using sucrose density gradient
<ol>
	<li>Preparation
	<ol>
		<li>Make 200 mL of TE buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA [pH 8.0]). Cool it by placing it on ice.</li>
		<li>Make 100 mL of 60% (w/v) sucrose solution in TE buffer by dissolving 60 g of sucrose in TE buffer, bringing the final volume to 100 mL. Cool it by placing it on ice.</li>
		<li>Make 6 mL of 40% (w/v) sucrose solution by adding 4 mL of 60% sucrose to 2 mL of TE buffer. Cool it by placing it on ice.</li>
		<li>Make 6 mL of 25% (w/v) sucrose solution by adding 2.5 mL of 60% sucrose to 3.5 mL of TE buffer. Cool it by placing it on ice.</li>
		<li>Make 4 mL of 10% (w/v) sucrose solution by adding 0.7 mL of 60% sucrose to 3.3 mL of TE buffer. Cool it by placing it on ice.</li>
		<li>Make 10 mL of protease and phosphatase inhibitor solution by adding one tablet of both protease and phosphatase inhibitors to 10 mL of TE buffer.</li>
		<li>Prepare tissue homogenate buffer by adding 4 mL of protease and phosphatase inhibitor solution to 50 mL of 60% sucrose stock solution. Cool it by placing it on ice.</li>
		<li>Prepare overlay buffer by adding 2 mL of protease and phosphatase inhibitor solution to 50 mL of TE buffer. Cool it by placing it on ice.</li>
		<li>Prepare 100 mL of 1x PBS by diluting 10 mL of 10x PBS in 90 mL of ultrapure water. Cool it by placing it on ice.</li>
		<li>Prepare 10 mL of 5% PGPE by diluting 500 &#181;L of PGPE in 9.5 mL of ultrapure water. Use a 1 mL syringe to measure out PGPE as it is viscous. Cool it by placing it on ice.</li>
		<li>Cool the following centrifuge tubes by placing them on ice: 1.7 mL microcentrifuge tubes, 15 mL centrifuge tubes, 50 mL high-speed centrifuge tubes, and 13 mL ultracentrifuge tubes.</li>
		<li>Cool the required centrifuges to 4 &#176;C: a microcentrifuge, a high-capacity centrifuge (for 15 mL tubes), a high-speed centrifuge (for 50 mL tubes), and an ultracentrifuge.</li>
	</ol>
	</li>
	<li>Tissue preparation
	<ol>
		<li>Use steps 1.2.1&#8211;1.2.9 to prepare the tissue for homogenization.</li>
		<li>Add 7 mL of cold tissue homogenate buffer and homogenize it on ice by moving the pestle up and down 20x. During the first five strokes, ensure the pestle goes to the bottom of the homogenizer.</li>
		<li>Transfer the homogenate to a cold 15 mL centrifuge tube.</li>
		<li>Rinse the homogenizer with 1 mL of cold tissue homogenate buffer and transfer it to the previously used 15 mL centrifuge tube.</li>
		<li>Bring the volume of the homogenate up to 10 mL with tissue homogenate buffer.</li>
		<li>Centrifuge the sample in a high-capacity centrifuge at 1,000 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer 8 mL of supernatant to a 50 mL centrifuge tube.</li>
		<li>Transfer 100 &#181;L of the remaining supernatant as PNS sample for purity analysis.</li>
	</ol>
	</li>
	<li>Sucrose gradient
	<ol>
		<li>Tilt the 50 mL centrifuge tube containing the 8 mL of PNS at a 45&#176; angle. Carefully add 6 mL of 40% sucrose solution, ensuring the two phases stay separate.</li>
		<li>In a similar fashion, slowly add 6 mL of 25% sucrose solution; then, add 4 mL of 10% sucrose solution; finally, add 8 mL of overlay buffer.</li>
		<li>Centrifuge the sample in a high-speed centrifuge at 30,000 x g at 4 &#176;C for 30 min.</li>
		<li>Transfer the top layer containing the LDs to a 1.7 mL microcentrifuge tube.</li>
		<li>For LD washing, follow the steps in section 1.4.</li>
	</ol>
	</li>
</ol>

<ol>
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Dr. Carr receives research support from Intercept Pharmaceuticals.

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 ...

rapid-lipid-droplet-isolation-protocol-using-well-established

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.1K Views.  Perelman School of Medicine, University of Pennsylvania. This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

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

Murine Pancreatic Islet Isolation

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 ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

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.