This work was supported by American Heart Association award 13SDG14500046 to P.D., a Sustainable Energy Education &#38; Research Center Award (Univ. of Tennessee) to P.D., and by the Physicians&#8217; Medical Education and Research Foundation (Univ. of Tennessee) award to J.M. The authors thank Caroline Leplante (Yale Univ.) for the protocol for converting fission yeast to spheroplasts; Eric T. Boder (Univ. of Tennessee) for the use of his shaking incubators, tabletop centrifuge, and Western Blot analysis equipment; and the Center for Environmental Biotechnology (Univ. Tennessee) for the use of their ultra-centrifuge; G&#252;nther Daum for yeast antibodies (Graz Univ. of Technology, Austria); the personnel of the Department of Obstetrics and Gynecology (Univ. Tennessee Medical Center) for technical assistance.

1. Isolating Lipid Droplets from (Fission) Yeast Cells
Isolation of droplets from the popular model organism budding yeast Saccharomyces cerevisiae is almost identical to the following protocol6. Differences in the preparations are noted.
1.&#160;Growing yeast cells
<ol>
	<li>Prepare the media. Combine 36 g&#160;of YE5S powder per liter of dH2O in glass bottles or culture flasks. About 2 L&#160;of media will be needed. Autoclave the media at 121 &#176;C for 20 min. Allow the media to cool to room temperature. For S. cerevisiae replace YE5S with YPD.</li>
	<li>Place 10-20 ml of the cooled YE5S media into a 250&#160;ml culture flask using sterile techniques. Inoculate the media with a small amount of yeast cells from an agar plate using a sterile wooden stick or equivalent. Let the cells grow to the desired optical density at 30 &#176;C. Measure the optical density at a wavelength of 595 nm using a spectrophotometer.</li>
	<li>Place the remaining media in the 2.8&#160;L flasks. Use no more than 1 L of media per 2.8&#160;L flask to ensure proper mixing during cell growth in the shaking incubator. The final yield of wet cells should be about 10 g&#160;to be able to acquire enough lipid droplets for proteomic and lipidomic analysis.</li>
	<li>Introduce the cells from step 1.2 into the 2.8&#160;L flasks each having 1 L of media that were prepared in step 1.1.</li>
	<li>Grow the cells to the desired density at 30 &#176;C.</li>
	<li>Make sure that the cells have lipid droplets. Reserve 1 ml of the cells. Measure the optical density (O.D.) of the sample. Add 0.1 ml of a 100 mM stock of BODIPY 493/503 in ethanol per O.D. of cells to the sample. Place 3 &#956;l&#160;of the sample between a glass slide and a glass cover slip. Visualize under a fluorescent microscope with a GFP filter (Figure 1A).</li>
	<li>Pellet the cells from step 1.5 at 4 &#176;C at 3,800 x g for 10 min. Work in batches depending on the capacity of the centrifuge tubes.</li>
	<li>Pour off the YE5S media and wash the cells with a buffer of EMM + 600 mM sorbitol. Sorbitol provides osmotic support.</li>
	<li>Transfer the cells to a sterile 50&#160;ml&#160;centrifuge tube and pellet the cells at 4 &#176;C at 2,000 x g for 10 min. Remove the supernatant. Weigh the wet cells. Resuspend the cells in 2 ml of (EMM + 600 mM sorbitol) buffer per gram of cells.</li>
</ol>
2.&#160;Converting fission yeast cells into spheroplasts and subsequent lysis
<ol>
	<li>Add 5 mg each of 1) yeast lytic enzyme and 2) yeast lysing enzymes from Trichoderma harzianum per gram of wet cells that were weighed in step 1.9. For S. cerevisiae augment these enzymes with the same amount of Zymolyase.</li>
	<li>Incubate the cells at 30 &#176;C under gentle shaking at 100 rpm for 60 min.</li>
	<li>Check to make sure that the spheroplasts have lipid droplets. By repeating step 1.6 (Figure 1B).</li>
</ol>
3.&#160;Density gradient centrifugation
<ol>
	<li>Pellet the spheroplasts by centrifugation at 4 &#176;C at 2,000 x g for 5 min.</li>
	<li>Carefully remove the supernatant using a plastic transfer pipette.</li>
	<li>Gently resuspend the pelleted spheroplasts with ice cold 10 mM Tris-HCl, 600 mM sorbitol, and 200 &#956;M EDTA. Keep in mind that the spheroplasts are very fragile.</li>
	<li>Pellet the spheroplasts again using the same settings as in step 3.1. Carefully remove the supernatant and resuspend the spheroplasts using a plastic transfer pipette in 12% Ficoll, 10 mM Tris-HCl, and 200 &#956;M EDTA at a concentration of 2 ml/g-cells.</li>
	<li>Transfer the resuspended spheroplasts to a glass homogenizer and gently homogenize on ice, 20-30 strokes, with the loose fitting pestle of the glass homogenizer.</li>
	<li>Transfer the lysed spheroplasts to centrifuge tubes and overlay with same volume of 12% Ficoll, 10 mM Tris-HCl, and 200 &#956;M EDTA buffer. Choose the number of centrifuge tubes to use so that each tube is about two-thirds full.</li>
	<li>Centrifuge at 4 &#176;C at 100,000 x g for 1.5 hr&#160;in an SW28 rotor or equivalent. Allow rotor to coast to a stop (rotor deceleration = 0).</li>
	<li>Gently transfer the top floating white layer (Figure 2A), which contains the droplets, to a new centrifuge tube(s) using either a pipettor or a bent Pasteur pipette. Add enough volume of 12% Ficoll, 10 mM Tris-HCl, and 200 &#956;M EDTA buffer to the sample so that it fills roughly one-third of the centrifuge tube.</li>
	<li>Add the equivalent volume of 8% Ficoll, 10 mM Tris-HCl, and 200 &#956;M EDTA buffer to the top of the sample(s) in the new centrifuge tube(s). After addition of this buffer the tubes should be about two-thirds full.</li>
	<li>Centrifuge at 4 &#176;C at 100,000 x g for 1 hr in an SW28 rotor or equivalent. Allow rotor to coast to a stop (rotor deceleration = 0).</li>
	<li>Gently transfer the top floating white layer (Figure 2B), which will contain the droplets, to a new centrifuge tube(s) using either a pipettor or a bent Pasteur pipette. Add 600 mM sorbitol, 8% Ficoll, 10 mM Tris-HCl, and 200 &#956;M EDTA buffer to the sample so that it fills about one-third of the centrifuge tube.</li>
	<li>Add the equivalent volume of 250 mM sorbitol, 10 mM Tris-HCl, and 200 &#956;M EDTA buffer to the top of the sample(s) in the new centrifuge tube(s). After addition of this buffer the tubes should be about two-thirds full.</li>
	<li>Centrifuge at 4 &#176;C at 100,000 x g for 1 hr in an SW28 rotor or equivalent. Allow rotor to coast to a stop (rotor deceleration = 0).</li>
	<li>Transfer top white layer (Figure 2C), which should contain only the lipid droplets and bound proteins, to dialysis tubing, and dialyze O/N into 10 mM Tris-HCl and 200 &#956;M EDTA buffer to eliminate the Ficoll.</li>
</ol>
2.&#160;Isolating Lipid Droplets from Human Placental Villous Cells
Placentas were collected from healthy women with singleton pregnancies undergoing elective cesarean section delivery prior to the onset of spontaneous labor at term. Subjects gave written, informed consent for the collection of their placenta. The collection, and subsequent use, of placentas was performed with approval from the University of Tennessee and University of Tennessee Graduate School of Medicine in Knoxville Institutional Review Board (#8757B and #3338, respectively).
1.&#160;Isolating human placental villous cells
Part 1 of the protocol is a modification of an earlier published protocol by Petroff et al.26
Prepare following solutions:
<ul>
	<li>NaCl (1 L): dissolve 9 g NaCl (M 58.4 g/mol) in dH2O to yield a 1 L solution. Filter sterilize (0.2&#160;&#956;m membrane).</li>
	<li>10x&#160;Hank&#8217;s balanced salt solution (10x&#160;HBSS) (1 L): 4 g KCl (M 74.5 g/mol); 0.6 g KH2PO4 (M 136.086 g/mol); 80 g NaCl (M 58.44 g/mol); Na2HPO4 (M 141.96 g/mol); 10 g D-glucose (M 180.16 g/mol). Dissolve each of the constituents in dH2O to yield a 1 L solution. Filter sterilize (0.2&#160;&#956;m membrane).</li>
	<li>Enzyme digestion buffer (0.6 ml): combine 70 ml 10x&#160;HBSS with 3.3 ml 7.5% Na Biscarbonate, 17.5 ml 1 M HEPES, and 266.1 ml dH2O; filter sterilize (0.2&#160;&#956;m membrane). Store at 4 &#176;C.</li>
	<li>DNase I: just before use, add 5 ml sterile 0.9% NaCl to a vial of DNase. Keep on ice until use or store at -20 &#176;C.</li>
</ul>
<ol>
	<li>Preparing placenta for dissection
	<ol>
		<li>Obtain a human placenta and process as close to the time of delivery as possible. Use a sealed vessel such as a cooler to transport the placenta. Always use caution when handling human biological materials.</li>
		<li>In the biological safety hood, transfer the placenta to a sterile autoclavable container, and carefully wash blood from placenta and membranes using sterile 0.9% NaCl (saline) solution. Discard bloody saline in the 1&#160;L beaker as liquid biological waste.</li>
		<li>Place the placenta on a sterile field, with the smooth surface bearing the umbilical cord facing up. With sharp, fine-point scissors and forceps remove fetal membranes and umbilical cord.</li>
		<li>Flip the placenta over so that the maternal surface (rough surface) is facing up. Remove the overlying basal plate tissue, about 3 mm from the surface.</li>
	</ol>
	</li>
	<li>Dissecting the placenta
	<ol>
		<li>Dissect one cotyledon at a time avoiding the chorionic plate. Collect villous tissue into a 250&#160;ml beaker with saline.</li>
		<li>Rinse tissue several times in a separate beaker with 0.9% NaCl by swirling with forceps, using the 1&#160;L beaker for liquid waste.</li>
		<li>Transfer one cotyledon at a time to a 150&#160;mm Petri dish. Hold with forceps and scrape tissue with razor blade from vessels on Petri dish. Place scraped tissue into separate beaker containing 0.9% NaCl. Repeat for all tissue pieces.</li>
		<li>Transfer small portion of the tissue to the cell dissociation sieve and rinse with 0.9% NaCl extensively until the eluate becomes clear. Repeat for all the tissue.</li>
		<li>After rinsing the scraped tissue in the cell dissociation sieve, drain it of excess liquid and weight. 
		At this point the tissue may be stored O/N&#160;in a sterile Erlenmeyer flask at 4 &#176;C in DMEM.</li>
	</ol>
	</li>
	<li>Enzymatic digestion of the placental tissue
	<ol>
		<li>Prepare the tissue digestion mixture: 100 ml of prewarmed enzyme dilution buffer, 1 ml DNase I and 20 ml 2.5% 10x&#160;trypsin.</li>
		<li>Divide the tissue, transferring 60 g from total collected tissue (usually about 250-300 g) to a 500&#160;ml sterile Erlenmeyer flask.</li>
		<li>Add the tissue digestion mixture to the Erlenmeyer flask containing the tissue, and incubate at 37 &#176;C in a shaker for 45 min at 150 rpm.</li>
	</ol>
	</li>
	<li>Collecting the dissociated cells
	<ol>
		<li>After the first 45 min dissociation, set the digestion flask at a tilt until tissue settles.</li>
		<li>Collect the supernatant, taking care not to collect undissociated tissue. Add an equal amount of HBSS to the collected supernatant and transfer into sterile 15&#160;ml centrifuge tubes. For additional portions of undissociated tissue, repeat procedure starting from step 3.2&#160;with the undissociated tissue.</li>
		<li>Centrifuge the homogenate at 4 &#176;C at 1,000 x g for 15 min.</li>
		<li>For each batch, following centrifugation, aspirate the supernatant without disturbing the pellet. The placental villous cells are predominantly in the white portion of the pellet, overlying the red blood cells.</li>
		<li>Transfer the villous cells (white portion of the pellet) to a sterile 50&#160;ml conical centrifuge tube and keep on ice.</li>
		<li>After collecting the cells from all three digestion stages, filter the suspension using a 100&#160;&#956;m Nylon cell strainer inserted in the top of a sterile 50&#160;ml conical centrifuge tube. If the filtration of the cell suspension slows, lift upward on the filter to draw a vacuum within the tube.</li>
		<li>Centrifuge at 4 &#176;C at 1,000 x g for 10 min. Carefully remove supernatant without disturbing the pellet.</li>
	</ol>
	</li>
</ol>
2. Homogenizing placental villous cells
<ol>
	<li>Before starting with the isolation process, prepare 50 ml of Hypotonic Lysis Medium (HLM) containing 20 mM Tris-HCl, pH 7.4 and 1 mM EDTA. Add protease inhibitors to HLM just before use and keep the medium on ice.</li>
	<li>In the biological safety hood, add 4 times the cell pellet volume of ice-cold HLM to the cells. Gently and thoroughly resuspend the cells by pipetting the cells up-and-down using a 10&#160;ml pipette.</li>
	<li>Incubate the suspended cells on ice for 10 min.</li>
	<li>Transfer the cells to the Dounce homogenizer, which should be on ice. Slowly homogenize the cells by applying 20-25 gentle strokes with the loose-fitting pestle of the homogenizer.</li>
	<li>Centrifuge the cell lysate at 4 &#176;C at 3,000 x g for 10 min to remove unbroken cells, cellular debris and nuclei.</li>
	<li>Collect the supernatant and transfer to a SW28&#160;tube (or alternative that can be centrifuged at 25,000 x g). Centrifuge at 4 &#176;C at 25,000 x g for 20 min to remove the mitochondria.</li>
</ol>
3. Isolating lipid droplets by ultracentrifugation
<ol>
	<li>Prepare 50 ml of 100 mM sodium carbonate (M 106 g/mol) buffer (pH 11.5) and 50 ml of 60% sucrose (w/w) solution. Add protease inhibitors to the sodium carbonate buffer just before use and keep on ice.</li>
	<li>Collect the supernatant from step 2.6 into a 50&#160;ml centrifuge tube, adjust to 20% of sucrose and transfer into the bottom of an ultracentrifuge tube for an SW28 rotor, or equivalent. Overlay density adjusted supernatant with ~10 ml of ice-cold 100 mM sodium carbonate buffer (pH 11.5) and 0.5-1 ml of ice-cold HLM to fill the tube.</li>
	<li>Centrifuge at 4 &#176;C at 130,000 x g for 45 min using SW28 swinging bucket rotor. Allow rotor to coast to a stop (rotor deceleration = 0).</li>
	<li>Collect the floating layer, adjust to 10% sucrose and transfer into the bottom of a 13.2&#160;ml ultracentrifuge tube for an SW41Ti rotor, or equivalent. Overlay density adjusted supernatant with about 5 ml of ice-cold 100 mM sodium carbonate buffer (pH 11.5) and 0.5 ml of ice-cold HLM.</li>
	<li>Centrifuge at 4 &#176;C at 274,000 x g for 60 min using SW41Ti swinging bucket rotor. Allow rotor to coast to a stop (rotor deceleration = 0) to minimize disruption of the lipid droplet layer.</li>
	<li>Carefully collect the top floating layer (Figure 2D) containing lipid droplets with a 1&#160;ml pipette and repeat step 3.4.</li>
	<li>Centrifuge at 4 &#176;C at 274,000 x g for 30 min using SW41Ti swinging bucket rotor. Allow rotor to coast to a stop (rotor deceleration = 0).</li>
	<li>Carefully collect the top white-colored floating layer containing lipid droplets with a bent Pasteur pipette into three 1.5&#160;ml tubes containing 100 &#956;l HLM.</li>
</ol>
Characterizing lipid droplets fraction (Protocols 1 and 2)
The recovery and purity of the lipid droplet fraction can be verified by Western Blot combined with light or electron microscopy. In addition, aliquots after different centrifugation steps can be collected and retained for determining purification efficiency. In Western Blotting, it is more appropriate to compare a volume of the lipid droplet fraction that represents an equivalent of the whole cell lysate than comparing lipid droplets to other membrane fractions on the basis of total protein content24.

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mouse+Prp19p+as+a+lipid+droplet-associated+protein+and+its+possible+involvement+in+the+biogenesis+of+lipid+droplets.">Identification of mouse Prp19p as a lipid droplet-associated protein and its possible involvement in the biogenesis of lipid droplets.</a> J. Biol. Chem. 282, 2456-2465 (2007).</li><li>Ding, Y. B., Wu, Y. B., Zeng, R., Liao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+profiling+of+lipid+droplet-associated+proteins+in+primary+adipocytes+of+normal+and+obese+mouse.">Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse.</a> Biochim. Biophys. Acta. 44, 394-406 (2012).</li><li>Jagerstrom, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+interact+with+mitochondria+using+SNAP23.">Lipid droplets interact with mitochondria using SNAP23.</a> Cell Biol. Int. 33, 934-940 (2009).</li><li>Xu, N. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FATP1-DGAT2+complex+facilitates+lipid+droplet+expansion+at+the+ER-lipid+droplet+interface.">The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface.</a> J. Cell Biol. 198, 895-911 (2012).</li><li>Ozeki, S., Cheng, J. L., Tauchi-Sato, K., Hatano, N., Taniguchi, H., Fujimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab18+localizes+to+lipid+droplets+and+induces+their+close+apposition+to+the+endoplasmic+reticulum-derived+membrane.">Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane.</a> J. Cell Sci. 118, 2601-2611 (2005).</li><li>Yang, H. Y., Galea, A., Sytnyk, V., Crossley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+size+of+lipid+droplets:+lipid+and+protein+factors.">Controlling the size of lipid droplets: lipid and protein factors.</a> Curr. Opin. Cell Biol. 24, 509-516 (2012).</li><li>Jacquier, N., Choudhary, V., Mari, M., Toulmay, A., Reggiori, F., Schneiter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+are+functionally+connected+to+the+endoplasmic+reticulum+in+Saccharomyces+cerevisiae.">Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.</a> J. Cell Sci. 124, 2424-2437 (2011).</li><li>Pu, J., Ha, C. W., Zhang, S., Jung, J. P., Huh, W. K., Liu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactomic+study+on+interaction+between+lipid+droplets+and+mitochondria.">Interactomic study on interaction between lipid droplets and mitochondria.</a> Protein Cell. 2, 487-496 (2011).</li><li>Shaw, C. S., Jones, D. A., Wagemakers, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+distribution+of+mitochondria+and+lipid+droplets+in+human+muscle+fibres.">Network distribution of mitochondria and lipid droplets in human muscle fibres.</a> Histochem. Cell Biol. 129, 65-72 (2008).</li><li>Goodman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gregarious+lipid+droplet.">The gregarious lipid droplet.</a> J. Biol. Chem. 283, 28005-28009 (2008).</li><li>Brasaemle, D. L., Wolins, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+of+fat:+an+evolving+model+of+lipid+droplet+assembly+and+expansion.">Packaging of fat: an evolving model of lipid droplet assembly and expansion.</a> J. Biol. Chem. 287, 2273-2279 (2012).</li></ol>

The authors declare no competing financial interests.

Critical steps within this protocol
Make sure to be consistent with media and cell densities during growth of cultured cells. Cellular lipid droplets are unique in that their associated proteins are highly dependent upon the environment in which the cells are cultured17. Therefore, the media in which the cells are grown and the density of the cells should be closely monitored before lysis.
The protein composition of lipid...

Cellular lipid droplets are dynamic organelles that serve multiple functions in cells. They are storage hubs for neutral lipids, which can be converted into energy or used for phospholipid synthesis. The droplets play central roles in physiological and pathological conditions including atherosclerosis, obesity and related metabolic diseases, and also infectious diseases1,2. In addition, they are intriguing sources for biodiesel fuels.

Much information on the cellular roles of lipid droplets has been obtained from proteomic and lipidomic analysis of droplets purified from wide-ranging organisms3. These organisms have included bacteria4,5, yeast6-11, plants12,13, nematodes14, and flies15,16. Given the interest in the role of lipid droplets in human metabolic diseases, droplets have also been isolated from cultured animal cells and animal tissues. Cultured cell lines have included 3T3-L1 adipocytes17, Chinese hamster ovary (CHO) K2 cells18, human hepatocyes19,20, and epithelial cell lines21. Animal tissues from which droplets have been isolated have included mouse skeletal muscle22, liver23, and mammary glands23. As mentioned above, the goal of most droplet isolation studies is to perform proteomic analysis on the bound factors and lipidomic analysis on the neutral and phospholipids.
Since neutral lipids - the most numerous component of lipid droplets - are less dense than most other cellular materials, isolation of droplets has traditionally been performed using density gradient centrifugation. That technique is the centerpiece of both preps presented here. Previous techniques6,24&#160;are combined and modified into a visual presentation of the isolation of droplets from cultured fission yeast cells and noncultured human cells obtained from placental tissue. The goal is to show the broad applicability of this technique by choosing two vastly different cell types as starting points for the droplet isolation. This technique should be useful for those wishing to isolate droplets from most organisms.
Protocol 1 describes the isolation of lipid droplets from the fission yeast, Schizosaccharomyces pombe, which has been used as a model for observing droplet formation during eukaryotic cell division25. The budding yeast Saccharomyces cerevisiae has been used extensively as a model organism for studying lipid droplet biology. Protocol 1 is applicable to both organisms and differences in the preparations are highlighted.&#160;&#160;&#160;

Protocol 2 describes the isolation of lipid droplets from placental villous cells, which are in turn obtained from human term placentas. The collection of term placentas provides a unique opportunity to safely and ethically obtain 200-250 g&#160;of readily available human tissue26, which contains significant numbers of lipid droplets. This is in contrast to most lipid droplet isolation work in higher eukaryotes where the droplets originate from cultured cells. In those studies, fatty acids are often added to the culture to promote the synthesis of neutral lipids and thus the growth of droplets. This is in contrast to the work here where lipid droplets are formed under native conditions in placental tissue.&#160;&#160;&#160;

The purities of the lipid droplet fractions are determined by Western Blot analysis using organelle marker antibodies. These two protocols will yield lipid droplet fractions that are suitable for subsequent proteomic and lipidomic analysis.

<table><tbody><tr><td>&lt;strong&gt;Protocol 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1. Growing yeast cells and converting to spheroplasts&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Edinburgh Minimal Media (EMM)</td><td>Sunrise Science Products</td><td>2005</td><td></td></tr><tr><td>Yeast extract with 5 supplements (YE5S)</td><td>Sunrise Science Products</td><td>2011</td><td>YE5S media with 225 mg/ml of each supplement: adeninie, histidine, leucine, lysine, uracil. The equivalent for budding yeast would be YPD.</td></tr><tr><td>YPD powder</td><td>Sunrise Science Products</td><td>1875</td><td>For &lt;i&gt;S. cerevisiae&lt;/i&gt;</td></tr><tr><td>Sorbitol</td><td>Fisher Scientific</td><td>BP439</td><td></td></tr><tr><td>Yeast Lytic Enzyme</td><td>MP Biomedicals</td><td>215352610</td><td></td></tr><tr><td>Lysing Enzymes from Trichoderma harzianum</td><td>Sigma-Aldrich</td><td>L1412</td><td></td></tr><tr><td>Zymolayse-20T</td><td>Sunrise Science Products</td><td>N0766391</td><td>For &lt;i&gt;S. cerevisiae&lt;/i&gt;</td></tr><tr><td>BODIPY 493/503</td><td>Invitrogen</td><td>D-3922</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-544-7</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Plastic transfer pipette</td><td>Fisher Scientific</td><td>137115AM</td><td></td></tr><tr><td>1 L glass bottle</td><td></td><td></td><td></td></tr><tr><td>250 ml flask</td><td></td><td></td><td></td></tr><tr><td>2.8 L flasks</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2. Yeast lipid droplet isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris-HCl</td><td>Fisher Scientific</td><td>BP153</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP120</td><td></td></tr><tr><td>Ficoll 400</td><td>Fisher Scientific</td><td>BP525</td><td></td></tr><tr><td>12-14k Spectra/Por Dialysis Membrane</td><td>SpectrumLabs</td><td>132680</td><td></td></tr><tr><td>EDTA-free Protease Inhibitor Cocktail Tablets</td><td>Roche Diagnostics</td><td>11873580001</td><td>irritant</td></tr><tr><td>Dounce Homogenizer&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D9938</td><td></td></tr><tr><td>Ultracentrifuge Tubes 25 mm x 89 mm (for SW28)</td><td>Beckman-Coulter</td><td>355642</td><td></td></tr><tr><td>12-14k Spectra/Por Dialysis Membrane</td><td>SpectrumLabs</td><td>132680</td><td></td></tr><tr><td>Temperature-controlled shaker</td><td>New Brunswick Scientific</td><td>C25KC</td><td></td></tr><tr><td>Thermo Sorvall Legend XTR centrifuge</td><td>Thermo-Scientific</td><td>75004521</td><td></td></tr><tr><td>Swinging Bucket Centrifuge Rotor</td><td>Thermo-Scientific</td><td>75003607</td><td></td></tr><tr><td>Fiberlite* F15-6x100y Fixed-Angle Rotor</td><td>Thermo-Scientific</td><td>75003698</td><td></td></tr><tr><td>Ultracentrifuge LB-M</td><td>Beckman-Coulter</td><td></td><td></td></tr><tr><td>SW28 Ultracentrifuge Rotor</td><td>Beckman-Coulter</td><td>342204</td><td></td></tr><tr><td>&lt;strong&gt;Protocol 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1. Placental villous cells isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable underpads</td><td>Fisher Scientific</td><td>23666062</td><td></td></tr><tr><td>Autoclavable pan (container), 3 L</td><td>Fisher Scientific</td><td>1336110</td><td></td></tr><tr><td>Fine scissors, sharp-sharp, straight</td><td>Fine Science Tools</td><td>1406011</td><td></td></tr><tr><td>London Forceps</td><td>Fine Science Tools</td><td>1108002</td><td></td></tr><tr><td>Dumont #7b Forceps</td><td>Fine Science Tools</td><td>1127020</td><td></td></tr><tr><td>Razor blades</td><td>Fisher Scientific</td><td>S65921</td><td></td></tr><tr><td>Screen cup for CD-1</td><td>Fisher Scientific</td><td>S1145</td><td></td></tr><tr><td>40 mesh screen&amp;nbsp;</td><td>Fisher Scientific</td><td>S0770</td><td></td></tr><tr><td>Fisherbrand cell stainers 100 &amp;mu;m</td><td>Fisher Scientific</td><td>22363549</td><td></td></tr><tr><td>150 mm Petri Dishes</td><td>Fisher Scientific</td><td>NC9054771</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S642</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P333</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P386</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310</td><td></td></tr><tr><td>2.5% Trypsin 10x</td><td>Invitrogen</td><td>15090046</td><td></td></tr><tr><td>DNase I grade II, from bovine pancreas</td><td>Roche Applied Science</td><td>10104159001</td><td></td></tr><tr><td>Sodium bicarbonate solution</td><td>Sigma-Aldrich</td><td>S8761</td><td></td></tr><tr><td>500 ml Erlenmeyer flasks</td><td></td><td></td><td></td></tr><tr><td>250 ml Beakers</td><td></td><td></td><td></td></tr><tr><td>15 ml Centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>10 ml Serological pipettes</td><td></td><td></td><td></td></tr><tr><td>50 ml Centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>11965084</td><td></td></tr><tr><td>&lt;strong&gt;2. Lipid droplets isolation from villous placental cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris-HCl</td><td>Fisher Scientific</td><td>BP153</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP120</td><td></td></tr><tr><td>D-Sucrose</td><td>Fisher Scientific</td><td>BP220</td><td></td></tr><tr><td>Sodium Carbonate&amp;nbsp;</td><td>Fisher Scientific</td><td>BP357</td><td></td></tr><tr><td>EDTA-free protease inhibitor cocktail tablets</td><td>Roche Diagnostics</td><td>11873580001</td><td>irritant</td></tr><tr><td>Dounce homogenizer&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D9938</td><td></td></tr><tr><td>Ultracentrifuge tubes 25 mm x 89 mm (for SW28)</td><td>Beckman-Coulter</td><td>355642</td><td></td></tr><tr><td>Ultra-Clear centrifuge tubes 14 mm x 89 mm (for SW41)</td><td>Beckman-Coulter</td><td>344059</td><td></td></tr><tr><td>Disposable borosilicate glass Pasteur pipets</td><td>Fisher Scientific</td><td>1367820C</td><td></td></tr><tr><td>Biological safety hood&amp;nbsp;</td><td>Thermo-Scientific</td><td></td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Temperature-controlled shaker</td><td>New Brunswick Scientific</td><td>C25KC</td><td></td></tr><tr><td>Thermo Sorvall Legend XTR centrifuge</td><td>Thermo-Scientific</td><td>75004521</td><td></td></tr><tr><td>Swinging Bucket Centrifuge Rotor</td><td>Thermo-Scientific</td><td>75003607</td><td></td></tr><tr><td>Ultracentrifuge LB-M</td><td>Beckman-Coulter</td><td></td><td></td></tr><tr><td>SW28 Ultracentrifuge Rotor</td><td>Beckman-Coulter</td><td>342204</td><td></td></tr><tr><td>SW41 Ti Ultracentrifuge Rotor</td><td>Beckman-Coulter</td><td>331336</td><td></td></tr><tr><td>&lt;strong&gt;Western blot&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IRDye 680 Goat Anti-Rabbit IgG</td><td>LI-COR</td><td>926-68071</td><td>dilution 1:15,000</td></tr><tr><td>IRDye 800CW Goat Anti-Mouse IgG</td><td>LI-COR</td><td>926-32210</td><td>dilution 1:5,000</td></tr><tr><td>NuPAGE Novex 12% Bis-Tris gels</td><td>Invitrogen</td><td>NP0341</td><td></td></tr><tr><td>&lt;strong&gt;Primary antibodies for Protocol 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Erg6p</td><td>gift from Dr. G. Daum</td><td>Graz University of Technology, Austria</td><td>dilution 1:5,000</td></tr><tr><td>Dpm1p</td><td>Abcam</td><td>ab113686</td><td>4 &amp;mu;g/ml</td></tr><tr><td>Por1p</td><td>gift from Dr. G. Daum</td><td>Graz University of Technology, Austria</td><td>dilution 1:5,000</td></tr><tr><td>Pma1p</td><td>gift from Dr. G. Daum</td><td>Graz University of Technology, Austria</td><td>dilution 1:10,000</td></tr><tr><td>Vma1p (anti-ATP6V1A)</td><td>Abcam</td><td>ab113745</td><td>0.5 &amp;mu;g/ml</td></tr><tr><td>&lt;strong&gt;Primary antibodies for Protocol 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Perilipin 2 (anti-ADFP)</td><td>Abcam</td><td>ab52355</td><td>2 &amp;mu;g/ml</td></tr><tr><td>Calnexin</td><td>Cell Signaling Technology</td><td>2679</td><td>dilution 1:1,000</td></tr><tr><td>GM130</td><td>Biorbyt</td><td>orb40533</td><td>dilution 1:25</td></tr><tr><td>COX IV</td><td>Cell Signaling Technology</td><td>4850</td><td>dilution 1:1,000</td></tr><tr><td>MEK1</td><td>Biorbyt</td><td>orb38775</td><td>dilution 1:50</td></tr></tbody></table>

isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

If the density gradient centrifugation worked as expected, the floating layer should contain lipid droplets and be depleted of other organelles throughout the progression of the high-speed spins.
For Protocol 1, Western Blots were performed with marker antibodies to lipid droplets (Erg6p), and organelles that have been found to interact with lipid droplets in yeast, ER (Dpm1p), mitochondria (Por1p), plasma membrane (Pma1p), and vacuoles (Vma1p).
Equal volumes of the...

Watch this Scientific Journal Video about Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas at JoVE.com

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

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

isolation-cellular-lipid-droplets-two-purification-techniques

Research

JoVE Journal

Bioengineering

19.6K Views.  University of Tennessee. Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

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

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Biochemistry

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Genetics

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Biology

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Immunology and Infection

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.

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 ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they are believed to serve a central role in the adaptation and remodeling of the thylakoid membrane under rapidly changing environmental conditions. The capacity to isolate plastoglobules of high purity has greatly facilitated their study through proteomic, lipidomic, and other methodologies. With plastoglobules of high purity and yield, it is possible to investigate their lipid and protein composition, enzymatic activity, and protein topology, among other possible molecular characteristics. This article presents a rapid and effective protocol for the isolation of plastoglobules from chloroplasts of plant leaf tissue and presents methodological variations for the isolation of plastoglobules and related lipid droplet structures from maize leaves, the desiccated leaf tissue of the resurrection plant, Eragrostis nindensis, and the cyanobacterium, Synechocystis sp. PCC 6803. Isolation relies on the low density of these lipid-rich particles, which facilitates their purification by sucrose density flotation. This methodology will prove valuable in the study of plastoglobules from diverse species.

A fast and efficient protocol is presented for the isolation of plastoglobule lipid droplets associated with various photosynthetic organisms. The successful preparation of isolated plastoglobules is a crucial first step that precedes detailed molecular investigations such as proteomic and lipidomic analyses.

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they ...

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

Summary

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Chapters in this video

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

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

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria