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.

<ol>
	<li>Ploegh, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lipid-based+model+for+the+creation+of+an+escape+hatch+from+the+endoplasmic+reticulum.">A lipid-based model for the creation of an escape hatch from the endoplasmic reticulum.</a> Nature. 448, 435-438 (2007).</li><li>Herker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+hepatitis+C+virus+particle+formation+requires+diacylglycerol+acyltransferase-1.">Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1.</a> Nat. Med. 16, 1295-1298 (2010).</li><li>Ding, Y. F., 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=Isolating+lipid+droplets+from+multiple+species.">Isolating lipid droplets from multiple species.</a> Nat. Protoc. 8, 43-51 (2013).</li><li>Kalscheuer, R., 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=Preparative+isolation+of+lipid+inclusions+from+Rhodococcus+opacus+and+Rhodococcus+rubber+and+identification+of+granule-associated+proteins.">Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus rubber and identification of granule-associated proteins.</a> Arch. Microbiol. 177, 20-28 (2001).</li><li>Low, K. L., 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+droplet-associated+proteins+are+involved+in+the+biosynthesis+and+hydrolysis+of+triacylglycerol+in+mycobacterium+bovis+bacillus+calmette-guerin.">Lipid droplet-associated proteins are involved in the biosynthesis and hydrolysis of triacylglycerol in mycobacterium bovis bacillus calmette-guerin.</a> J. Biol. Chem. 285, 21662-21670 (2010).</li><li>Leber, R., Zinser, E., Zellnig, G., Paltauf, F., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+lipid+particles+of+the+yeast,+Saccharomyces+cerevisiae.">Characterization of lipid particles of the yeast, Saccharomyces cerevisiae.</a> Yeast. 10, 1421-1428 (1994).</li><li>Grillitsch, K., 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+particles/droplets+of+the+yeast+Saccharomyces+cerevisiae+revisitied:+lipidome+meets+proteome.">Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisitied: lipidome meets proteome.</a> Biochim. Biophys. Acta. , 1165-1176 (2011).</li><li>Binns, D., 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=An+intimate+collaboration+between+peroxisomes+and+lipid+bodies.">An intimate collaboration between peroxisomes and lipid bodies.</a> J. Cell Biol. 173, 719-731 (2006).</li><li>Ivashov, V. A., 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=Lipidome+and+proteome+of+lipid+droplets+from+the+methylotropic+yeast+Pichia+pastoris.">Lipidome and proteome of lipid droplets from the methylotropic yeast Pichia pastoris.</a> Biochim. Biophys. Acta. 1831, 282-290 (2013).</li><li>Connerth, M., Grillitsch, K., Kofeler, H., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+lipid+particles+from+yeast.">Analysis of lipid particles from yeast.</a> Lipidomics: Vol. 1: Methods and Protocols. 579, 359-374 (2009).</li><li>Wolinski, H., Kohlwein, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+analysis+of+lipid+droplet+metabolism+and+dynamics+in+yeast.">Microscopic analysis of lipid droplet metabolism and dynamics in yeast.</a> Methods Mol. Biol. 457, 151-163 (2008).</li><li>Jolivet, P., 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=Protein+composition+of+oil+bodies+in+Arabidopsis+thaliana+ecotype+WS.">Protein composition of oil bodies in Arabidopsis thaliana ecotype WS.</a> Plant Physiol. Biochem. 42, 501-509 (2004).</li><li>Katavic, V., Agrawal, G. K., Hajduch, M., Harris, S. L., Thelen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+and+lipid+composition+analysis+of+oil+bodies+from+two+Brassica+napus+cultivers.">Protein and lipid composition analysis of oil bodies from two Brassica napus cultivers.</a> Proteomics. 6, 4586-4598 (2006).</li><li>Zhang, P., 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=Proteomic+study+and+marker+protein+identification+of+Caenorhabditis+elegans+lipid+droplets.">Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets.</a> Mol. Cell. Proteomics. 11, 317-328 (2012).</li><li>Krahmer, N., Hilger, M., Kory, N., Wilfling, F., Stoehr, G., Mann, M., Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+correlation+profiles+identify+lipid+droplet+proteins+with+high+confidence.">Protein correlation profiles identify lipid droplet proteins with high confidence.</a> Mol. Cell Proteomics. 12, 1115-1126 (2013).</li><li>Cermelli, S., Gou, Y., Gross, S. P., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipid-droplet+proteome+reveals+that+droplets+are+a+protein-storage+depot.">The lipid-droplet proteome reveals that droplets are a protein-storage depot.</a> Curr. Biol. 16, 1783-1795 (2006).</li><li>Brasaemle, D. L., Dolios, G., Shapiro, L., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+proteins+associated+with+lipid+droplets+of+basal+and+lipolytically+stimulated+3T3-L1+adipocytes.">Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.</a> J. Biol. Chem. 279, 46835-46842 (2004).</li><li>Liu, P. S., Ying, Y. S., Zhao, Y. M., Mundy, D. I., Zhu, M. F., Anderson, R. G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chinese+hamster+ovary+K2+cell+lipid+droplets+appear+to+be+metabolic+organelles+involved+in+membrane+traffic.">Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic.</a> J. Biol. Chem. 279, 3787-3792 (2004).</li><li>Fujimoto, 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+major+proteins+in+the+lipid+droplet-enriched+fraction+isolated+from+the+human+hepatocyte+cell+line+HuH7.">Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.</a> Biochim. Biophys. Acta. , 47-59 (2004).</li><li>Sato, 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=Proteomic+profiling+of+lipid+droplet+proteins+in+hepatoma+cell+lines+expressing+hepatitis+C+virus+core+protein.">Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.</a> J. Biochem. 139, 921-930 (2006).</li><li>Umlauf, E., Csaszar, E., Moertelmaier, M., Schuetz, G. J., Parton, R. G., Prohaska, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+stomatin+with+lipid+bodies.">Association of stomatin with lipid bodies.</a> J. Biol. Chem. 279, 23699-23709 (2004).</li><li>Zhang, H. N., 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=Proteome+of+skeletal+muscle+lipid+droplet+reveals+association+with+mitochondria+and+apolipoprotein+A-I.">Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein A-I.</a> J. Proteome Res. 10, 4757-4768 (2011).</li><li>Wu, C. C., Howell, K. E., Neville, M. C., Yates, J. R., McManaman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+reveal+a+link+between+the+endoplasmic+reticulum+and+lipid+secretory+mechanisms+in+mammary+epithelial+cells.">Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells.</a> Electrophoresis. 21, 3470-3482 (2000).</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=Isolation+of+lipid+droplets+from+cells+by+density+gradient+centrifugation.">Isolation of lipid droplets from cells by density gradient centrifugation.</a> Curr. Protoc. Cell Biol. , 3.15.1-3.15.12 (2005).</li><li>Long, A. P., 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+droplet+de+novo+formation+and+fission+are+linked+to+the+cell+cycle+in+fission+yeast.">Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast.</a> Traffic. 13, 705-714 (2012).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods Mol. Med. 121, 203-217 (2006).</li><li>Cho, S. 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 border="0" cellpadding="0" cellspacing="0" width="802">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PROTOCOL #1:&#160;</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.Growing yeast cells and converting to spheroplasts</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Edinburgh Minimal Media (EMM)</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">2005</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;">Yeast extract with 5 supplements (YE5S)</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">2011</td>
			<td style="width:167px;">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 height="20">
			<td height="20" style="height:20px;">YPD powder</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">1875</td>
			<td>For S. cerevisiae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP439</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Lytic Enzyme</td>
			<td>MP Biomedicals</td>
			<td style="width:119px;">215352610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysing Enzymes from Trichoderma harzianum</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">L1412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zymolayse-20T</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">N0766391</td>
			<td>For S. cerevisiae&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BODIPY 493/503</td>
			<td>Invitrogen</td>
			<td style="width:119px;">D-3922</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">12-544-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">12-542-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic transfer pipette</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">137115AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 liter glass bottle</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 ml flask</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.8 liter flasks</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2. Yeast lipid droplet isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll 400</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-14k Spectra/Por Dialysis Membrane</td>
			<td>SpectrumLabs</td>
			<td style="width:119px;">132680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free Protease Inhibitor Cocktail Tablets</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11873580001</td>
			<td>irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce Homogenizer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Tubes 25x89mm (for SW28)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">355642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-14k Spectra/Por Dialysis Membrane</td>
			<td>SpectrumLabs</td>
			<td style="width:119px;">132680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Temperature-controlled shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;">C25KC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Sorvall Legend XTR centrifuge</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75004521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swinging Bucket Centrifuge Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiberlite* F15-6x100y Fixed-Angle Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROTOCOL #2&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1. Placental villous cells isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable underpads</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">23666062</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Autoclavable pan (container), 3L</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">1336110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine scissors, sharp-sharp, straight</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1406011</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">London Forceps</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1108002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #7b Forceps</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1127020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor blades</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S65921</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screen cup for CD-1</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S1145</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 mesh screen&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S0770</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand cell stainers 100&#956;m</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">22363549</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150 mm Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">NC9054771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S642</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">P333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">P386</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S374</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">D16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.5% trypsin 10x</td>
			<td>Invitrogen</td>
			<td style="width:119px;">15090046</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I grade II, from bovine pancreas</td>
			<td>Roche Applied Science</td>
			<td style="width:119px;">10104159001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium bicarbonate solution</td>
			<td>Sigma-aldrich</td>
			<td style="width:119px;">S8761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">500 ml Erlenmeyer flasks</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">250 ml beakers</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">15 ml centrifuge tubes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">10 ml serological pipettes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50 ml centrifuge tubes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">DMEM</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">11965084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2. Lipid droplets isolation from villous placental cells</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Sucrose</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Carbonate&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11873580001</td>
			<td>irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce homogenizer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge tubes 25x89mm (for SW28)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">355642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-Clear centrifuge tubes 14x89mm (for SW41)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable borosilicate glass pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">1367820C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Biological safety hood&#160;</td>
			<td style="width:163px;">Thermo-Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterbath</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Temperature-controlled shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;">C25KC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Sorvall Legend XTR centrifuge</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75004521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swinging Bucket Centrifuge Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW41 Ti Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Western blot</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">IRDye 680 Goat Anti-Rabbit IgG</td>
			<td style="width:163px;">LI-COR</td>
			<td style="width:119px;">926-68071</td>
			<td>dilution 1:15000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">IRDye&#160; 800CW Goat Anti-Mouse IgG</td>
			<td style="width:163px;">LI-COR</td>
			<td style="width:119px;">926-32210</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">NuPAGE&#174; Novex&#174; 12% Bis-Tris gels</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">NP0341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">primary antibodies for PROTOCOL #1</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Erg6p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Dpm1p</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab113686</td>
			<td>4 &#956;g/ml</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Por1p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Pma1p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Vma1p (anti-ATP6V1A)</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab113745</td>
			<td>0.5 &#956;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;"></td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">primary antibodies for PROTOCOL #2</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">perilipin 2 (anti-ADFP)</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab52355</td>
			<td>2 &#956;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">calnexin</td>
			<td style="width:163px;">Cell Signaling technology</td>
			<td style="width:119px;">2679</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">GM130</td>
			<td style="width:163px;">Biorbyt</td>
			<td style="width:119px;">orb40533</td>
			<td>dilution 1:25</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">COX IV</td>
			<td style="width:163px;">Cell Signaling technology</td>
			<td style="width:119px;">4850</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">MEK1</td>
			<td style="width:163px;">Biorbyt</td>
			<td style="width:119px;">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

19.5K 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

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...