Questo lavoro è stato supportato dal premio American Heart Association 13SDG14500046 al PD, a Educazione Energia Sostenibile &amp; Research Center Award (Univ. of Tennessee) al PD, e dal Physicians &#39;Medical Education and Research Foundation (Univ. of Tennessee) premio a JM L&#39; autori ringraziano Caroline Leplante (Yale University.) per il protocollo per la conversione di lievito di fissione a sferoplasti; Eric T. Boder (Univ. of Tennessee) per l&#39;utilizzo dei suoi incubatori stringono, tavolo centrifuga e apparecchiature Western Blot, e il Centro per Biotecnologie Ambientali (Univ. Tennessee) per l&#39;uso della loro ultra-centrifuga, Günther Daum per gli anticorpi di lievito (Graz University of Technology, Austria.), il personale del Dipartimento di Ostetricia e Ginecologia (Univ. Tennessee Medical Center) per l&#39;assistenza tecnica .

1. Isolare lipidi goccioline da (fissione) cellule di lievito Isolamento di goccioline dalla famosa organismo modello in erba lievito Saccharomyces cerevisiae è quasi identico al seguente protocollo 6. Le differenze tra le preparazioni sono noti. 1. Cellule di lievito in crescita <ol><li> Preparare il supporto. Unire 36 g di YE5S polvere per litro di dH 2 O in bottiglie di vetro o flaconi di coltura. Saranno necessari circa 2 L di media. Autoclavare il supporto a 121 ° C per 20 min. Lasciare i media a raffreddare a temperatura ambiente. Per S. cerevisiae sostituire YE5S con YPD. </li><li> Posizionare 10-20 ml del terreno YE5S raffreddati in un pallone di coltura da 250 ml con tecniche sterili. Inoculare il supporto con una piccola quantità di cellule di lievito da una piastra di agar usando un bastoncino di legno sterile o equivalente. Lasciare che le cellule crescono alla densità ottica desiderata a 30 ° C. Misurare la densità ottica allalunghezza d&#39;onda di 595 nm usando uno spettrofotometro. </li><li> Posizionare i mezzi rimasti in 2,8 L palloni. Utilizzare non più di 1 L di supporto per 2.8 L pallone per garantire la corretta miscelazione durante la crescita delle cellule in incubatore agitazione. La resa finale di cellule umide dovrebbe essere di circa 10 g per essere in grado di acquisire abbastanza goccioline lipidiche nell&#39;analisi proteomica e lipidomico. </li><li> Introdurre le cellule dal punto 1.2 in 2,8 L palloni ciascuno con 1 L di mezzi di comunicazione che sono stati preparati nel passaggio 1.1. </li><li> Crescere le cellule alla densità desiderata a 30 ° C. </li><li> Assicurarsi che le cellule hanno goccioline lipidiche. Riserva 1 ml delle cellule. Misurare la densità ottica (OD) del campione. Aggiungere 0,1 ml di uno stock di 100 mm BODIPY 493/503 in etanolo per OD delle cellule al campione. Posizionare 3 microlitri del campione tra un vetrino ed un vetrino di vetro. Visualizzare sotto un microscopio a fluorescenza con un filtro GFP (Figura 1A). </li><li> Agglomerare le cellule dal punto 1.5 a 4 &amp; #176; C a 3.800 xg per 10 min. Lavorare in lotti a seconda della capacità delle provette da centrifuga. </li><li> Versare i media YE5S e lavare le cellule con un buffer di EMM + 600 mm sorbitolo. Sorbitolo fornisce il supporto osmotica. </li><li> Trasferire le cellule ad una sterile 50 ml provetta da centrifuga e agglomerare le cellule a 4 ° C a 2.000 xg per 10 min. Rimuovere il surnatante. Pesare le celle umide. Risospendere le cellule in 2 ml di (EMM + 600 mM sorbitolo) tampone per grammo di cellule. </li></ol> 2. Conversione di cellule di lievito di fissione in sferoplasti e successiva lisi <ol><li> Aggiungere 5 mg ciascuna di 1) lievito litico enzima e 2) lievito lisi enzimi da Trichoderma harzianum per grammo di cellule umide che furono pesate nel passaggio 1.9. Per S. cerevisiae augment questi enzimi con la stessa quantità di Zymolyase. </li><li> Incubare le cellule a 30 ° C sotto leggera agitazione a 100 rpm per 60 min. </li><li> Verificare che le sferoplasti hanno dr lipidioplets. Ripetendo il punto 1.6 (Figura 1B). </li></ol> 3. Gradiente di densità centrifugazione <ol><li> Agglomerare le sferoplasti mediante centrifugazione a 4 ° C a 2.000 xg per 5 min. </li><li> Rimuovere con cautela il surnatante con una pipetta di trasferimento di plastica. </li><li> Risospendere delicatamente gli sferoplasti pellet con ghiaccio freddo 10 mM Tris-HCl, 600 mM sorbitolo e 200 mM EDTA. Tenete a mente che le sferoplasti sono molto fragili. </li><li> Pellet di nuovo le sferoplasti utilizzando le stesse impostazioni come al punto 3.1. Rimuovere con cura il supernatante e risospendere le sferoplasti utilizzando una pipetta di plastica a 12% Ficoll, 10 mM Tris-HCl, 200 mM e EDTA ad una concentrazione di 2 ml / g-cellule. </li><li> Trasferire i sferoplasti risospese ad un omogeneizzatore vetro e omogeneizzare dolcemente sul ghiaccio, 20-30 tratti, con il pestello larghi del omogeneizzatore vetro. </li><li> Trasferire i sferoplasti lisati di provette da centrifuga e sovrapporre con la stessa volume del 12% Ficoll, 10 mM Tris-HCl, 200 mM tampone EDTA. Scegliere il numero di provette da centrifuga da usare in modo che ogni tubo è di circa due terzi. </li><li> Centrifugare a 4 ° C a 100.000 xg per 1,5 ore in un rotore SW28 o equivalente. Lasciare rotore per inerzia (rotore di decelerazione = 0). </li><li> Trasferire delicatamente lo strato superiore galleggiante bianco (Figura 2A), che contiene le goccioline, in un tubo da centrifuga (s) utilizzando una pipetta o un piegato pipetta Pasteur. Aggiungere abbastanza volume del 12% Ficoll, 10 mM Tris-HCl, 200 mM e tampone EDTA al campione in modo da riempire circa un terzo della provetta da centrifuga. </li><li> Aggiungere il volume equivalente dell&#39;8% Ficoll, 10 mM Tris-HCl, 200 mM e tampone EDTA all&#39;inizio del campione (s) nella nuova provetta da centrifuga (s). Dopo l&#39;aggiunta di questo buffer tubi dovrebbero essere di circa due terzi pieno. </li><li> Centrifugare a 4 ° C a 100.000 xg per 1 ora in un rotore SW28 o equivalente. Lasciare rotore per inerzia (rotor decelerazione = 0). </li><li> Trasferire delicatamente la parte superiore bianca livello fluttuante (Figura 2B), che conterrà le goccioline, in un tubo da centrifuga (s) utilizzando una pipetta o un piegato pipetta Pasteur. Aggiungere 600 mM sorbitolo, 8% Ficoll, 10 mM Tris-HCl, 200 mM e tampone EDTA al campione in modo da riempire circa un terzo della provetta da centrifuga. </li><li> Aggiungere il volume equivalente di 250 mM sorbitolo, 10 mM Tris-HCl, e tampone 200 mM EDTA all&#39;inizio del campione (s) nella nuova provetta da centrifuga (s). Dopo l&#39;aggiunta di questo buffer tubi dovrebbero essere di circa due terzi pieno. </li><li> Centrifugare a 4 ° C a 100.000 xg per 1 ora in un rotore SW28 o equivalente. Lasciare rotore per inerzia (rotore di decelerazione = 0). </li><li> Trasferire top strato bianco (Figura 2C), che dovrebbe contenere solo le goccioline lipidiche e proteine ​​legate, a tubi di dialisi, e Dializzare O / N in 10 mM Tris-HCl e 200 mM tampone EDTA per eliminare il Ficoll. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Isolare lipidi goccioline da cellule umane placentare villi Placente sono stati raccolti da donne sane con gravidanza singola fase elettiva sezione parto cesareo prima del travaglio spontaneo a termine. I soggetti hanno dato, il consenso informato scritto per la raccolta dei loro placenta. La raccolta e successivo utilizzo, di placente è stata effettuata con l&#39;approvazione presso l&#39;Università del Tennessee e University of Tennessee Graduate School of Medicine di Knoxville Institutional Review Board (# 8757B e # 3338, rispettivamente). 1. Isolare le cellule dei villi placentari umane Parte 1 del protocollo è una modifica di un protocollo pubblicato prima da Petroff et al 26. Preparare le seguenti soluzioni: <ul><li> NaCl (1 L): sciogliere 9 g di NaCl (M 58,4 g / mol) in dH 2 O per fornire una soluzione di 1 L. Filtro sterilizzare (0,2; Micron membrana). </li><li> Soluzione salina bilanciata di 10x Hank (10x HBSS) (1 L): 4 g KCl (M 74,5 g / mol), 0,6 g KH 2 PO 4 (M 136,086 g / mol), 80 g di NaCl (M 58,44 g / mol); Na 2 HPO 4 (M 141.96 g / mol), 10 g di D-glucosio (M 180.16 g / mol). Sciogliere ciascuno dei componenti in dH 2 O per fornire una soluzione di 1 L. Sterilizzare Filter (0,2 micron membrana). </li><li> Tampone di digestione enzimatica (0,6 ml): unire 70 ml 10x HBSS con 3,3 ml di 7,5% Na Biscarbonate, 17,5 ml di 1 M HEPES, e 266,1 ml di dH 2 O e sterilizzare il filtro (0,2 micron membrana). Conservare a 4 ° C. </li><li> DNasi I: appena prima dell&#39;uso, aggiungere 5 ml sterile 0,9% NaCl ad una fiala di DNasi. Tenere in ghiaccio fino al momento dell&#39;uso o conservare a -20 ° C. </li></ul><ol><li> Preparazione placenta per la dissezione <ol><li> Ottenere una placenta e di processo più vicino possibile al momento della consegna del possibile umano. Utilizzare un vaso sigillato come un dispositivo di raffreddamento per trasportare la placenta. Usare sempre cautela quando la manomateriali biologici umani Ling. </li><li> Nella cappa di sicurezza biologica, trasferire la placenta in un contenitore autoclavabile sterile, e lavare accuratamente sangue dalla placenta e le membrane con una soluzione sterile allo 0,9% di NaCl (soluzione fisiologica). Gettare salina sanguinosa nel bicchiere 1 L come rifiuti biologici liquidi. </li><li> Posizionare la placenta su un campo sterile, con la superficie liscia recante il cordone ombelicale rivolto verso l&#39;alto. Con taglienti, forbici a punta fine e pinze rimuovere membrane fetali e cordone ombelicale. </li><li> Capovolgere la placenta in modo che la superficie materna (superficie ruvida) sia rivolta verso l&#39;alto. Rimuovere il tessuto sovrastante piastra basale, circa 3 mm dalla superficie. </li></ol></li><li> Sezionare la placenta <ol><li> Sezionare un cotiledone alla volta per evitare la piastra coriali. Raccogliere il tessuto dei villi in un bicchiere da 250 ml con soluzione fisiologica. </li><li> Sciacquare tessuto più volte in un bicchiere separato con 0,9% NaCl agitando con le pinze, con il bicchiere 1 L per rifiuti liquidi. </li><li> Trasferire un cotiledone alla volta per una piastra di Petri di 150 mm. Tenere con una pinza e raschiare il tessuto con lametta da navi in ​​piastra di Petri. Collocare il tessuto raschiato nel bicchiere separato contenente 0.9% NaCl. Ripetere l&#39;operazione per tutti i pezzi di tessuto. </li><li> Trasferire piccola porzione di tessuto al setaccio dissociazione cellulare e risciacquare con 0,9% NaCl ampiamente finché l&#39;eluato diventa chiaro. Ripetere l&#39;operazione per tutto il tessuto. </li><li> Dopo aver sciacquato il tessuto raschiato nel setaccio dissociazione cellulare, scolarla di liquidi in eccesso e peso. A questo punto il tessuto può essere conservato O / N in un matraccio di Erlenmeyer sterile a 4 ° C in DMEM. </li></ol></li><li> Digestione enzimatica del tessuto placentare <ol><li> Preparare il tessuto digestione impasto: 100 ml di preriscaldata tampone di diluizione enzimatica, 1 ml di DNasi I e 20 ml 2,5% 10x tripsina. </li><li> Dividere il tessuto, trasferendo 60 g di tessuto raccolti totale (solitamente circa 250-300 g) a una beuta sterile 500 ml. </li><li&gt; Aggiungere la miscela di digestione tessuto sulla beuta contenente il tessuto e incubare a 37 ° C in un agitatore per 45 min a 150 rpm. </li></ol></li><li> Raccogliere le cellule dissociate <ol><li> Dopo i primi 45 minuti di dissociazione, impostare il pallone digestione in una inclinazione fino tessuto si deposita. </li><li> Raccogliere il surnatante, facendo attenzione a non raccogliere il tessuto non dissociato. Aggiungere una quantità uguale di HBSS al supernatante raccolto e trasferire in provette da centrifuga da 15 ml sterili. Per ulteriori porzioni di tessuto non dissociato, ripetere la procedura dal punto 3.2 con il tessuto non dissociato. </li><li> Centrifugare l&#39;omogeneizzato a 4 ° C a 1000 xg per 15 min. </li><li> Per ciascun lotto, dopo la centrifugazione, aspirare il surnatante senza disturbare il pellet. Le cellule dei villi placentari sono prevalentemente nella porzione bianca del pellet, sormontante i globuli rossi. </li><li> Trasferire le cellule dei villi (parte bianca del pellet) per una sterile 50 ml tubo da centrifuga conica e tenere in ghiaccio. </li><li> Dopo aver raccolto le cellule da tutte e tre le fasi di digestione, filtrare la sospensione con una 100 um filtro cella Nylon inserita nella parte superiore di una sterile 50 ml provetta da centrifuga conica. Se la filtrazione della sospensione cellulare rallenta, sollevare verso l&#39;alto sul filtro per disegnare un vuoto all&#39;interno del tubo. </li><li> Centrifugare a 4 ° C a 1000 xg per 10 min. Rimuovere con attenzione il surnatante senza disturbare il pellet. </li></ol></li></ol> 2. Omogeneizzando le cellule dei villi placentari <ol><li> Prima di iniziare con il processo di isolamento, preparare 50 ml di ipotonico Lysis Medium (HLM) contenente 20 mM Tris-HCl, pH 7.4 e 1 mM EDTA. Aggiungere inibitori della proteasi per HLM appena prima dell&#39;uso e mantenere il mezzo su ghiaccio. </li><li> Nella cappa biologica, aggiungere 4 volte il volume pellet di cellule di HLM ghiacciata alle cellule. Delicatamente e completamente sospendere nuovamente le cellule pipettando le cellule su-und-giù con una pipetta 10 ml. </li><li> Incubare le cellule sospese in ghiaccio per 10 min. </li><li> Trasferire le cellule per l&#39;omogeneizzatore Dounce, che dovrebbe essere sul ghiaccio. Lentamente omogeneizzare le cellule applicando 20-25 movimenti delicati con il pestello larghi del omogeneizzatore. </li><li> Centrifugare il lisato cellulare a 4 ° C a 3000 xg per 10 minuti per rimuovere le cellule intatte, detriti e nuclei cellulari. </li><li> Raccogliere il surnatante e trasferire in una provetta SW28 (o alternativo che può essere centrifugato a 25.000 xg). Centrifugare a 4 ° C a 25.000 xg per 20 min per rimuovere i mitocondri. </li></ol> 3. Isolare gocce lipidiche da ultracentrifugazione <ol><li> Preparare 50 ml di 100 mM di carbonato di sodio (M 106 g / mol) tampone (pH 11,5) e 50 ml di soluzione di saccarosio al 60% (w / w). Aggiungere inibitori della proteasi al tampone carbonato di sodio appena prima dell&#39;uso e mantenere in ghiaccio. </li><li> Raccogliere il supernatante dal passo 2.6 in una provetta da centrifuga da 50 ml, regolareal 20% di saccarosio e trasferimento nel fondo di un tubo un&#39;ultracentrifuga per un rotore SW28, o equivalente. Surnatante densità Overlay regolato con ~ 10 ml di tampone carbonato di sodio 100 mM ghiacciato (pH 11,5) e 0,5-1 ml di ghiacciata HLM per riempire il tubo. </li><li> Centrifugare a 4 ° C a 130.000 xg per 45 minuti usando SW28 rotore oscillante. Lasciare rotore per inerzia (rotore di decelerazione = 0). </li><li> Raccogliere il livello fluttuante, regolare al 10% di saccarosio e trasferire nel fondo di un tubo 13,2 ml un&#39;ultracentrifuga per un rotore SW41Ti, o equivalente. Densità Overlay adeguato surnatante con circa 5 ml di tampone 100 mM di carbonato di sodio ghiacciato (pH 11,5) e 0,5 ml di HLM ghiacciata. </li><li> Centrifugare a 4 ° C a 274.000 xg per 60 minuti con SW41Ti oscillante rotore secchio. Lasciare rotore per inerzia (rotore di decelerazione = 0) per minimizzare i disagi dello strato lipidico gocciolina. </li><li> Raccogliere accuratamente il livello fluttuante superiore (Figura 2D) contenentegocce lipidiche con una pipetta 1 ml e ripetere il punto 3.4. </li><li> Centrifugare a 4 ° C a 274.000 xg per 30 minuti usando SW41Ti oscillante rotore secchio. Lasciare rotore per inerzia (rotore di decelerazione = 0). </li><li> Raccogliere accuratamente lo strato galleggiante di colore bianco-top contenenti goccioline lipidiche con un&#39;inclinazione pipetta Pasteur in tre provette da 1,5 ml contenenti 100 microlitri HLM. </li></ol> Frazione di goccioline lipidiche che caratterizzano (protocolli 1 e 2) Il recupero e la purezza della frazione lipidica gocciolina possono essere verificate mediante western blot combinata con la luce o la microscopia elettronica. Inoltre, aliquote dopo differenti fasi di centrifugazione possono essere raccolti e conservati per determinare l&#39;efficienza di depurazione. In Western Blotting, è più appropriato per confrontare un volume della frazione lipidica gocciolina che rappresenta un equivalente di tutta lisato cellulare che confrontare goccioline lipidiche ad altre frazioni di membrana su tegli base del contenuto totale di proteine ​​24.

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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>

Gli autori dichiarano interessi finanziari concorrenti.

<html> Passaggi critici all&#39;interno di questo protocollo Assicurarsi coerente con media e densità cellulari durante la crescita delle cellule in coltura. Goccioline lipidiche cellulari sono unici in quanto le loro proteine ​​associate sono altamente dipendenti l&#39;ambiente in cui le cellule sono coltivate 17. Pertanto, il supporto in cui si coltivano le cellule e la densità delle cellule devono essere strettamente monitorati prima lisi....

Gocce lipidiche cellulari sono organelli dinamiche che servono molteplici funzioni nelle cellule. Essi sono hub stoccaggio dei lipidi neutri, che possono essere convertiti in energia o utilizzati per la sintesi dei fosfolipidi. Le goccioline giocano un ruolo centrale in condizioni fisiologiche e patologiche tra cui l&#39;aterosclerosi, obesità e malattie metaboliche, malattie infettive e anche 1,2. Inoltre, sono fonti interessanti per carburanti biodiesel. Molte informazioni sui ruoli cellulari di gocce lipidiche è stato ottenuto da analisi proteomica e lipidomico di goccioline purificate da organismi di ampio respiro 3. Questi organismi hanno incluso i batteri, lieviti 4,5 6-11, piante 12,13, nematodi 14, e vola 15,16. Dato l&#39;interesse per il ruolo di goccioline lipidiche in malattie metaboliche umane, goccioline sono anche stati isolati da cellule animali in coltura etessuti Nimal. Linee cellulari coltivate hanno incluso 3T3-L1 adipociti 17, ovaia di criceto cinese (CHO), cellule K2 18, hepatocyes umane 19,20, e linee cellulari epiteliali 21. Tessuti animali da cui sono state isolate gocce hanno incluso il mouse muscolo scheletrico 22, 23 fegato e ghiandole mammarie 23. Come accennato in precedenza, l&#39;obiettivo della maggior parte degli studi isolamento gocciolina è effettuare analisi proteomica sui fattori legati e analisi lipidomico sul neutro e fosfolipidi. Poiché i lipidi neutri - i più numerosi dei componenti di goccioline lipidiche - sono meno dense maggior parte degli altri materiali cellulari, isolamento di gocce è stata tradizionalmente eseguita utilizzando gradiente di densità centrifugazione. Tale tecnica è il fulcro di entrambi preparazioni presentate qui. Tecniche precedenti 6,24 sono combinate e modificate in una presentazione visiva dell&#39;isolamento di goccioline da cellule di lievito di fissione coltivatee cellule umane noncultured ottenute dal tessuto placentare. L&#39;obiettivo è di mostrare la vasta applicabilità di questa tecnica scegliendo due tipi di cellule molto diverse come punti per l&#39;isolamento gocciolina di partenza. Questa tecnica dovrebbe essere utile per coloro che desiderano isolare le goccioline dalla maggior parte degli organismi. Protocollo 1 descrive l&#39;isolamento di goccioline lipidiche dal lievito di fissione, Schizosaccharomyces pombe, che è stato usato come modello per osservare la formazione di goccioline durante la divisione cellulare eucariotica 25. Il lievito Saccharomyces cerevisiae in erba è stato ampiamente utilizzato come organismo modello per lo studio dei lipidi droplet biologia. Protocollo 1 è applicabile a entrambi gli organismi e le differenze nei preparativi sono evidenziati. Protocollo 2 descrive l&#39;isolamento di goccioline lipidiche dalle cellule dei villi placentari, che sono a loro volta ottenuti da placenta umana termine. Ilraccolta di placente termine offre un&#39;opportunità unica di ottenere in modo sicuro ed eticamente 200-250 g di facilmente reperibile tessuto umano 26, che contiene un numero significativo di goccioline lipidiche. Questo è in contrasto con la maggior parte del lavoro di isolamento lipidi gocciolina in eucarioti superiori dove le goccioline originano da cellule coltivate. In questi studi, gli acidi grassi sono spesso aggiunti alla coltura per promuovere la sintesi di lipidi neutri e quindi la crescita di goccioline. Questo è in contrasto con il lavoro qui dove goccioline lipidiche si formano in condizioni native nel tessuto placentare. Le purezza delle frazioni lipidi goccioline sono determinati mediante analisi Western Blot usando anticorpi marcatori organelli. Questi due protocolli produrranno frazioni lipidiche goccioline che sono adatti per la successiva analisi proteomica e lipidomico.

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			<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>
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		<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

Gocce lipidiche sono organelli dinamiche che si possono trovare nella maggior parte delle cellule procariote eucariotiche e certe. Strutturalmente, le goccioline costituiti da un nucleo di lipidi neutri circondato da un monostrato di fosfolipidi. Una delle tecniche più utili nella determinazione dei ruoli cellulari delle goccioline è stato individuazione proteomica di proteine ​​legate, che possono essere isolati con le goccioline. Qui, due metodi sono descritti per isolare gocce lipidiche e le loro proteine ​​legate da due ampie eucarioti: fissione lievito e cellule dei villi placentari umani. Anche se entrambe le tecniche hanno differenze, il metodo principale - Densità di centrifugazione in gradiente - è condivisa da entrambi i preparativi. Questo dimostra l&#39;ampia applicabilità delle tecniche di isolamento droplet presentate. Nel primo protocollo, le cellule di lievito vengono convertiti in sferoplasti mediante digestione enzimatica delle loro pareti cellulari. I sferoplasti risultanti sono poi gently lisato in un omogeneizzatore larghi. Ficoll viene aggiunto al lisato di fornire un gradiente di densità, e la miscela viene centrifugata per tre volte. Dopo la prima rotazione, le goccioline lipidiche sono localizzati al livello fluttuante colore bianco delle provette da centrifuga con il reticolo endoplasmatico (ER), la membrana plasmatica, e vacuoli. Due successivi giri sono utilizzati per rimuovere questi altri tre organelli. Il risultato è uno strato che ha solo goccioline e proteine ​​legate. Nel secondo protocollo, le cellule dei villi placentari sono isolate da placenta umana termine per digestione enzimatica con tripsina e DNasi I. Le cellule vengono omogeneizzati in un omogeneizzatore larghi. Passaggi a bassa velocità e centrifugazione a velocità media vengono utilizzati per rimuovere le cellule continue, detriti cellulari, nuclei e mitocondri. Il saccarosio è aggiunto l&#39;omogeneizzato per fornire un gradiente di densità e la miscela viene centrifugata per separare le goccioline lipidiche dall&#39;altro cellulafrazioni r. La purezza delle goccioline lipidiche in entrambi i protocolli è confermata mediante analisi Western Blot. Le frazioni di gocce di entrambe le preparazioni sono adatti per la successiva analisi proteomica e lipidomico.

Se la centrifugazione in gradiente di densità lavorato come previsto, il livello fluttuante dovrebbe contenere goccioline lipidiche e essere impoverito di altri organelli durante la progressione delle rotazioni ad alta velocità. Per il protocollo 1, Western Blot sono stati eseguiti con anticorpi marcatori a gocce lipidiche (Erg6p), e gli organelli che sono stati trovati ad interagire con le goccioline di lipidi nel lievito, ER (Dpm1p), mitocondri (Por1p), membrana plasmatica (Pma1p), e vac...

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

Due tecniche per isolare gocce lipidiche cellulari da 1) cellule di lievito e 2) placente umane sono presentati. Il fulcro di entrambe le procedure è la densità centrifugazione in gradiente, dove lo strato risultante galleggiante contenente le goccioline può essere facilmente visualizzato occhio, estratto, e quantificato mediante analisi Western Blot per la purezza.

Isolamento dei lipidi cellulari Goccioline: due tecniche di purificazione partire da cellule di lievito e placente umane

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.  Due tecniche per isolare gocce lipidiche cellulari da 1) cellule di lievito e 2) placente umane sono presentati. Il fulcro di entrambe le procedure è la densità centrifugazione in gradiente, dove lo strato risultante galleggiante contenente le goccioline può essere facilmente visualizzato occhio, estratto, e quantificato mediante analisi Western Blot per la purezza. 

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.

Rotazione Cultura sistemi cellulari per colture cellulari umane: cellule del trofoblasto umano come modello

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

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Bioingegneria

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).

Estrazione dei lipidi cellulari per analisi mirate Stabile diluizione isotopica cromatografia liquida-spettrometria di massa

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.

Sviluppo, di espansione, e<em&gt; In vivo</em&gt; Monitoraggio delle cellule NK umane da cellule staminali embrionali umane (hESC) ed e cellule staminali pluripotenti indotte (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.

Doppio strato lipidico delle vescicole Generation Uso Microfluidic getto

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.

Isolamento di primarie cellule del colon tumorali umane dai tessuti chirurgici e Coltura direttamente su più elastica e flessibile substrati per trazione citometria

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.

Tecniche per l&#39;evoluzione della robusta lievito Pentoso-fermentazione per bioconversione della lignocellulosa a etanolo

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.

Isolamento di cellule staminali mesenchimali da periostio alveolare umano e gli effetti della vitamina D su attività osteogenica delle cellule derivate dal periostio

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.

Assorbimento di nuove nanoparticelle rivestite con lipidi contenenti Falcarindiol di cellule staminali mesenchimali umane

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.

Isolamento di fibroblasti dermici umani adulti dalla pelle addominale e generazione di cellule staminali pluripotenti indotte utilizzando un metodo non integrato

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.

Misurazione del potenziale d'azione ottico a singola cellula in cardiomiociti derivati da cellule staminali pluripotenti indotte umane

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