Name: Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit (Video) | JoVE Journal
Uploaded: 2019-04-19T14:00:00.000+00:00
Duration: 8 min 43 s
Description: Learn a rapid lipid droplet isolation method for liver disease research using mouse liver tissue. This modified kit protocol enables simultaneous purification of multiple organelles with comparable purity to traditional sucrose gradient methods.

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5 minutes

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><li>Brasaemle, D. L., Wolins, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+Lipid+Droplets+from+Cells+by+Density+Gradient+Centrifugation%5BTitle%5D)+AND+%22Current+Protocols+in+Cell+Biology%22%5BJournal%5D)">Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation.</a> Current Protocols in Cell Biology. 72, (2016).</li>
<li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolating+lipid+droplets+from+multiple+species%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Isolating lipid droplets from multiple species.</a> Nature Protocols. 8 (1), 43-51 (2013).</li>
<li>Gao, Q., Goodman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+lipid+droplet-a+well-connected+organelle%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">The lipid droplet-a well-connected organelle.</a> Frontiers in Cell and Developmental Biology. 3, 49(2015).</li>
<li>Correnti, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethanol+and+C2+ceramide+activate+fatty+acid+oxidation+in+human+hepatoma+cells%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Ethanol and C2 ceramide activate fatty acid oxidation in human hepatoma cells.</a> Scientific Reports. 8 (1), 12923(2018).</li>
<li>Williams, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+role+for+ceramide+synthase+6+in+mouse+and+human+alcoholic+steatosis%5BTitle%5D)+AND+%22FASEB+Journal%22%5BJournal%5D)">A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.</a> FASEB Journal. 32 (1), 130-142 (2018).</li>
<li>Carr, R. M., Ahima, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathophysiology+of+lipid+droplet+proteins+in+liver+diseases%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Pathophysiology of lipid droplet proteins in liver diseases.</a> Experimental Cell Research. 340 (2), 187-192 (2016).</li>
<li>Carr, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perilipin+Staining+Distinguishes+Between+Steatosis+and+Nonalcoholic%5BTitle%5D)+AND+%22Steatohepatitis+in+Adults+and+Children.+Clinical+Gastroenterology+and+Hepatology%22%5BJournal%5D)">Perilipin Staining Distinguishes Between Steatosis and Nonalcoholic.</a> Steatohepatitis in Adults and Children. Clinical Gastroenterology and Hepatology. 15 (1), 145-147 (2017).</li>
<li>Carr, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduction+of+TIP47+improves+hepatic+steatosis+and+glucose+homeostasis+in+mice%5BTitle%5D)+AND+%22American+Journal+of+Physiology%3A+Regulatory%2C+Integrative+and+Comparative+Physiology%22%5BJournal%5D)">Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice.</a> American Journal of Physiology: Regulatory, Integrative and Comparative Physiology. 302 (8), R996-R1003 (2012).</li>
<li>Carr, R. M., Peralta, G., Yin, X., Ahima, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absence+of+perilipin+2+prevents+hepatic+steatosis%2C+glucose+intolerance+and+ceramide+accumulation+in+alcohol-fed+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Absence of perilipin 2 prevents hepatic steatosis, glucose intolerance and ceramide accumulation in alcohol-fed mice.</a> PLoS One. 9 (5), e97118(2014).</li>
<li>Tsai, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+constitutive+lipid+droplet+protein+PLIN2+regulates+autophagy+in+liver%5BTitle%5D)+AND+%22Autophagy%22%5BJournal%5D)">The constitutive lipid droplet protein PLIN2 regulates autophagy in liver.</a> Autophagy. 13 (7), 1130-1144 (2017).</li>
<li>Guo, Y., Cordes, K. R., Farese, R. V. Jr, Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipid+droplets+at+a+glance%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Lipid droplets at a glance.</a> Journal of Cell Science. 122 (Pt 6), 749-752 (2009).</li>
<li>Liu, K., Czaja, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+lipid+stores+and+metabolism+by+lipophagy%5BTitle%5D)+AND+%22Cell+Death+and+Differentiation%22%5BJournal%5D)">Regulation of lipid stores and metabolism by lipophagy.</a> Cell Death and Differentiation. 20 (1), 3-11 (2013).</li>
<li>Mannik, J., Meyers, A., Dalhaimer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+cellular+lipid+droplets%3A+two+purification+techniques+starting+from+yeast+cells+and+human+placentas%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas.</a> Journal of Visualized Experiments. (86), e509814(2014).</li>
<li>Sato, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+profiling+of+lipid+droplet+proteins+in+hepatoma+cell+lines+expressing+hepatitis+C+virus+core+protein%5BTitle%5D)+AND+%22Journal+of+Biochemistry%22%5BJournal%5D)">Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.</a> Journal of Biochemistry. 139 (5), 921-930 (2006).</li>
<li>Wu, C. C., Howell, K. E., Neville, M. C., Yates, J. R. 3rd, McManaman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomics+reveal+a+link+between+the+endoplasmic+reticulum+and+lipid+secretory+mechanisms+in+mammary+epithelial+cells%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells.</a> Electrophoresis. 21 (16), 3470-3482 (2000).</li>
<li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphologically+and+Functionally+Distinct+Lipid+Droplet+Subpopulations%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Morphologically and Functionally Distinct Lipid Droplet Subpopulations.</a> Scientific Reports. 6, 29539(2016).</li>
</ol>

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><tbody><tr><td>BioExpress Vortex Mixer</td><td>GeneMate</td><td>S-3200-1</td><td>Vortex Mixer</td></tr><tr><td>Centrifuge 54242 R</td><td>Eppendorf</td><td>54242 R</td><td>Refrigerated Counter Microcentrifuge&lt;br/&gt; Rotor: FA-45-24-11</td></tr><tr><td>Centrifuge Sorvall RC6 +</td><td>Thermo Scientific</td><td>RC6+</td><td>Refrigerated High Speed Centrifuge&lt;br/&gt; Rotor: F21S-8x50y</td></tr><tr><td>Centrifuge Sorvall Legend RT+</td><td>Thermo Scientific</td><td>Legend RT+</td><td>Refrigerated High Capacity Centrifuge&lt;br/&gt; Rotor: 75006445 Bucket: 75006441</td></tr><tr><td>cOmplete Protease Inhibitor Cocktail</td><td>Sigma Aldrich</td><td>04 693 116 001</td><td>Protease Inhibitor Tablets</td></tr><tr><td>Diagenode Bioruptur UCD-200</td><td>Diagenode</td><td>UCD-200</td><td>Sonication System</td></tr><tr><td>Dounce Tissue Grinder (45 mL)</td><td>Wheaton</td><td>357546</td><td>Use Loose pestle</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (1x)</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>Endoplasmic Reticulum Isolation Kit</td><td>Sigma Aldrich</td><td>ER0100</td><td>For ER kit Extraction:&lt;br/&gt; Contains:&lt;br/&gt; Isotonic Extraction Buffer 5X 100 mL&lt;br/&gt; Hypotonic Extraction Buffer 10 mL&lt;br/&gt; Calcium chloride, 2.5 M solution 5 mL&lt;br/&gt; OptiPrep Density Gradient Medium 100 mL&lt;br/&gt; Blunt Nosed Needle 1 ea.</td></tr><tr><td>External light source for flourescent excitation</td><td>Leica</td><td>Leica EL6000</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma Aldrich</td><td>H1758</td><td>Hydrochloric Acid</td></tr><tr><td>ImageJ</td><td></td><td></td><td>Image Analysis Software</td></tr><tr><td>Inverted Modulating Contrast Microscope</td><td>Leica</td><td>Leica DM IRB</td><td></td></tr><tr><td>Lysosome Enrichment Kit for Tissues and Cultured Cells</td><td>Thermo Fisher Scientific</td><td>89839</td><td>For Lysosome Isolation</td></tr><tr><td>Microscope Camera</td><td>Qimaging</td><td>QICAM fast 1394</td><td></td></tr><tr><td>Optima XL-100K Ultracentrifuge</td><td>Beckman-Coulter</td><td>XL-100K</td><td>Ultracentrifuge&lt;br/&gt; Rotor: SW41Ti</td></tr><tr><td>Pasteur Pipettes</td><td>Fisher Scientific</td><td>22-042817</td><td></td></tr><tr><td>Petri Dish 5 cm</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>PhosSTOP</td><td>Sigma Aldrich</td><td>04 906 845 001</td><td>Phosphatase Inhibior Tablets</td></tr><tr><td>Sterile Surgical Blade #10</td><td>Pioneer</td><td>S2646</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td>Crystalline/Certified ACS</td></tr><tr><td>Triglyceride Liquicolor Kit</td><td>Stanbio&amp;nbsp;</td><td>2200-225</td><td>Colorimetric Triglyceride kit</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td>For TE buffer</td></tr><tr><td>Triton X-100</td><td>Fisher BioReagents</td><td>BP151-100</td><td>5%, Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, Protein Lysis Reagent</td></tr><tr><td>UltraPure EDTA (0.5M, pH 8.0)</td><td>Thermo Fisher Scientific</td><td>15575-038</td><td>For TE buffer</td></tr></tbody></table>

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

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...

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

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

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Research

JoVE Journal

Biology

This video demonstrates a technique for visualizing the formation of the immunological synapse in natural killer (NK) cells on a supported lipid bilayer (SLB). The SLB is first labeled with a fluorophore-tagged NK cell activating receptor ligand, followed by incubation with NK cells. Subsequently, the cells are stained for polymerized actin and perforin-positive lytic granules. This approach enables the visualization of synapse formation using stimulated emission depletion (STED) microscopy.

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability.  We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

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Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

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Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

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SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

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Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

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Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Learn a rapid lipid droplet isolation method for liver disease research using mouse liver tissue. This modified kit protocol enables simultaneous purification of multiple organelles with comparable purity to traditional sucrose gradient methods.

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

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