这项工作是由美国心脏协会奖13SDG14500046帕金森病，可持续能源教育与研究中心奖（田纳西大学）PD的支持，并由医师的医学教育和研究基金会（田纳西大学）颁发给JM的作者感谢卡罗琳Leplante（耶鲁大学）的协议转换裂殖酵母原生质球;埃里克T. BODER（田纳西大学）为利用他颤抖孵化器，台式离心机，和Western blot分析设备;和中心环境生物技术（Univ.田纳西州）的使用他们的超离心机;半滑舌鳎道姆（科技，奥地利格拉茨大学）​​酵母抗体;妇产科（Univ.田纳西州医学中心）的技术援助部门的人员。

1。从（裂变）酵母细胞中分离脂滴芽殖酵母酿酒酵母从流行的模式生物滴的隔离几乎是相同的以下协议6。在筹备的差异说明。 1。培养酵母细胞<ol><li>准备媒体。在玻璃瓶或培养瓶中结合36克YE5S粉每升DH 2 O的。约2升的介质是必​​要的。高压灭菌介质在121℃下20分钟。允许介质冷却至室温。对于S。酵母与YPD代替YE5S。 </li><li>放置10-20冷却的YE5S媒体毫升到使用无菌技术的250ml培养瓶中。用少量的从琼脂板用无菌木棍或等效的酵母细胞接种的介质。让细胞生长至所需要的光学密度在30℃下测量在光密度波长的使用分光光度计595nm处。 </li><li>将剩余的介质在2.8升瓶。使用不超过1升介质每2.8升的烧瓶中，以确保细胞生长在振荡培养箱中适当混合。湿细胞的最终产量应为约10克至能够获得足够的脂滴的蛋白质和脂质组分析。 </li><li>引入来自步骤1.2将细胞放入2.8升锥形瓶中分别具有1升的制备步骤1.1的介质。 </li><li>生长的细胞所需的密度在30℃下</li><li>确保该细胞有脂滴。保留1毫升细胞。测量样品的光学密度（OD）。在每个细胞的OD乙醇加0.1毫升BODIPY五百〇三分之四百九十三的100mM储备到样品。放置3微升玻璃载玻片和盖玻片之间的样品。荧光显微镜用GFP过滤器（ 图1A）根据可视化。 </li><li>从步骤1.5颗粒的细胞在4＆＃176，C在3,800 xg离心10分钟。取决于离心管的容量分批工作。 </li><li>倒出YE5S媒体和EMM + 600 1mM的缓冲山梨醇洗涤细胞。山梨糖醇渗透提供支持。 </li><li>将细胞转移到无菌的50ml离心管中，沉淀细胞，在4℃以2000×g离心10分钟。除去上清液。称量湿细胞。重悬在2ml的每克（EMM + 600 mM的山梨糖醇）的缓冲单元的单元。 </li></ol> 2。裂殖酵母细胞转化为原生质球和随后的裂解<ol><li>每添加1 5毫克）酵母溶解酶和2）酵母裂解从木霉每克在步骤1.9 ​​称重的湿细胞的酶。对于S。酵母增强这些酶具有的Zymolyase相同量。 </li><li>孵育的细胞在30℃下温和振摇在100rpm下60分钟。 </li><li>检查以确保球状体具有脂质博士oplets。通过重复步骤1.6（ 图1B）。 </li></ol> 3。密度梯度离心<ol><li>通过离心在4℃下沉淀的原生质球在2000×g离心5分钟。 </li><li>小心地取下用塑料移液管的上清液。 </li><li>轻轻重悬沉淀的原生质球用冰冷的10mM的Tris-HCl，600mM的山梨醇和200μMEDTA中。请记住，球状体是很脆弱的。 </li><li>使用相同的设置步骤3.1再次沉淀的原生质球。小心地取出上清液，并用12％的Ficoll塑料移液管，10毫摩尔Tris-HCl和200μMEDTA以2毫升/克细胞的浓度重悬原生质球。 </li><li>转移的原生质球悬浮于玻璃匀浆器匀化轻轻置于冰上20-30笔画，用玻璃匀浆器的宽松杵。 </li><li>转移裂解原生质球到离心管中，并用覆盖相同的VO12％聚蔗糖，10毫摩尔Tris-HCl和200μMEDTA缓冲液的lume明。选择离心管的数量来使用，因此，每个管大约是三分之二满。 </li><li>离心机在4℃下以100,000×g离心1.5小时在SW28转子或等效物。允许转子惯性停止（转子减速= 0）。 </li><li>上方浮动的白色层（ 图2A），其中包含液滴，轻轻转移到使用一个移液管或弯曲的巴斯德吸管一个新的离心管中（次）。添加足够量的12％Ficoll制备，10毫摩尔Tris-HCl和200μMEDTA缓冲液的样品并使其填满大约三分之一的离心管中。 </li><li>添加8％聚蔗糖的等效体积的10mM Tris-HCl中，200μMEDTA缓冲液中的样品（多个）在新的离心管中（次）的顶部。除了这个缓冲后的管应约三分之二满。 </li><li>离心机在4℃下以100,000×g离心1小时，在SW28转子或等效物。允许转子惯性停止（ROTOR减速= 0）。 </li><li>轻轻转上漂浮的白色层（ 图2B），其中将包含液滴，以使用一个移液器或弯曲巴斯德吸管一个新的离心管（S）。添加600mM的山梨糖醇，8％的Ficoll，10毫摩尔Tris-HCl和200μMEDTA缓冲液的样品并使其填满大约三分之一的离心管。 </li><li>加250毫当量体积山梨醇，10毫摩尔Tris-HCl和200μMEDTA缓冲液中的样品（多个）在新的离心管中（次）的顶部。除了这个缓冲后的管应约三分之二满。 </li><li>离心机在4℃下以100,000×g离心1小时，在SW28转子或等效物。允许转子惯性停止（转子减速= 0）。 </li><li>传输时的白色层（ 图2C），这应该只包含脂滴和结合蛋白，透析管路，透析和O / N为10毫米的Tris-HCl和200微米EDTA缓冲液，以消除聚蔗糖。 </li></ol><p类=“jove_title”&gt; 2。从人胎盘绒毛细胞中分离脂滴胎盘是从健康妇女与单胎妊娠择期剖宫产分娩发作自然分娩的足月之前收集的。主题写了，因为他们的胎盘的收集知情同意书。胎盘的收集，以及随后的使用，是与来自田纳西州和医学田纳西研究生院的大学的大学在诺克斯维尔机构审查委员会（＃8757B和＃3338，分别）批准执行。 1。分离人胎盘绒毛细胞该协议的第1部分是较早发布的协议由佩特罗夫等人 26的修改准备以下解决方案： <ul><li>氯化钠（1升）：溶解9克氯化钠（M58.4克/摩尔）在DH 2 O，以产生1 L溶液。过滤消毒（0.2;微米膜）。 </li><li> 10倍的Hank平衡盐溶液（10X HBSS）（1升）：4克氯化钾（M74.5克/摩尔）;0.6克KH 2 PO 4（M136.086克/摩尔），80克氯化钠（M58.44克/摩尔）;的Na 2 HPO 4（M141.96克/摩尔）;10克D-葡萄糖（M180.16克/摩尔）。溶解各构成要素在DH 2 O，以产生1 L溶液。过滤消毒（0.2微米膜）。 </li><li>酶消化缓冲液（0.9毫升）：结合70毫升10倍的HBSS与3.3毫升7.5％的Na Biscarbonate，17.5毫升1 M羟乙基，和266.1毫升DH 2 O，过滤消毒（0.2微米膜）。贮存于4℃。 </li><li> DNA酶I：在使用前，加入5毫升无菌0.9％NaCl溶液，以DNA酶的小瓶。置于冰上直至使用或存放于-20°C。 </li></ul><ol><li>胎盘做准备解剖<ol><li>获得人胎盘和过程尽可能靠近输送尽可能的时间。使用密封容器，如冷却器运送胎盘。请务必小心，当手林志玲人体生物材料。 </li><li>在生物安全罩，胎盘转移到无菌高压灭菌的容器中，并用无菌0.9％氯化钠（食盐）溶液胎盘及胎膜仔细洗血。丢弃血性盐水中的1升烧杯中的液体生物废物。 </li><li>将胎盘在无菌区，与光滑表面轴承朝上脐带。有棱，细点剪刀和镊子取出胎膜和脐带。 </li><li>翻转胎盘过来，使产妇表面（粗糙面）朝上。去除上覆的基底板的组织，从表面约3mm。 </li></ol></li><li>解剖胎盘<ol><li>解剖一个子叶的时间避免绒毛膜板。收集绒毛组织置于250ml烧杯中的盐水。 </li><li>通过用钳子漩，使用1升烧杯中的液体废物冲洗组织几次在0.9％NaCl中的单独的烧杯中。 </li><li>在一次传输一个子叶至150毫米的培养皿。握住钳子和刮组织与从培养皿船只刀片。放置刮掉组织成含有0.9％NaCl的单独的烧杯中。重复所有的组织块。 </li><li>转移组织的小部分细胞解离筛，并用0.9％的NaCl冲洗广泛直至洗出液变清晰。重复所有的组织。 </li><li>漂洗刮下组织中的细胞解离筛后，沥干多余的液体和重量的它。 在这一点上，组织可以被存储O / N中的无菌锥形瓶中，在4℃在DMEM中。 </li></ol></li><li>胎盘组织的酶消化<ol><li>制备组织消化混合物：百毫升预热的酶稀释缓冲液，1毫升DNA酶I和20毫升2.5％的胰蛋白酶10倍。 </li><li>分裂组织，转移60克从总采集的组织（通常约250-300克），以500毫升的无菌锥形瓶中。 </li><lI&gt;添加的组织消化混合物含有该组织的锥形烧瓶中，并在摇床上以150rpm 45分钟孵育在37℃。 </li></ol></li><li>收集脱落的细胞<ol><li>第45分钟解离后，在倾斜设置的消化瓶中，直到组织落户。 </li><li>收集上清液，注意不要收集未解离的组织。添加HBSS等量收集到的上清液并转移到无菌的15ml离心管中。对于未解离组织的额外部分，重复从步骤3.2与未解离的组织开始。 </li><li>离心匀浆液在4℃下以1,000×g离心15分钟。 </li><li>对于每个批次，离心后，吸出上清液，而不会干扰沉淀。胎盘绒毛细胞是主要在沉淀的白色部分，上覆的红血细胞。 </li><li>转让绒毛细胞（颗粒的白色部分）到STerile 50ml锥形离心管中，并保持在冰上。 </li><li>从所有三个阶段消化收集细胞后，用无菌的50ml锥形离心管的顶部插入一个100微米尼龙细胞过滤器过滤悬浮液。如果细胞悬浮液的过滤变慢，向上抬起在过滤器上绘制在管内的真空度。 </li><li>离心机在4℃下以1,000×g离心10分钟。小心取出上清液，而不会干扰颗粒。 </li></ol></li></ol> 2。均质胎盘绒毛细胞<ol><li>首先是隔离过程之前，制备含20毫米的Tris-HCl，pH 7.4和1毫米EDTA50毫升低渗裂解中等（HLM）的。添加蛋白酶抑制剂，以HLM只是在使用前，保持介质上的冰。 </li><li>在生物安全罩中，添加冰冷HLM 4倍的细胞沉淀物体积的细胞。轻轻地，彻底地吹打细胞悬浮细胞向上的D-下，用10毫升吸管。 </li><li>在冰上孵育悬浮细胞10分钟。 </li><li>将细胞转移到Dounce匀浆，这应该是在冰上。慢慢地通过应用20-25温柔的笔触与均质的宽松杵同质化的细胞。 </li><li>离心细胞裂解物在4℃下以3,000×g离心10分钟以除去完整的细胞，细胞碎片和细胞核。 </li><li>收集上清液并转移至SW28管（或替代，可以在25,000 XG离心）。离心机在4℃下以25,000 xg离心20分钟以除去线粒体。 </li></ol> 3。通过离心分离脂滴<ol><li>制备50毫升100毫碳酸钠（M 106克/摩尔）缓冲液（pH 11.5）和50毫升60％蔗糖（w / w的）解决方案。添加蛋白酶抑制剂的碳酸钠缓冲液在使用前并保持在冰上。 </li><li>从步骤2.6收集上清液至50ml离心管中，调整到蔗糖和转移到超速离心管中的SW28转子，或等效的底部的20％。覆盖密度调节上清液用约10毫升冰冷的碳酸盐100mM的钠缓冲液（pH 11.5）和0.5-1毫升冰冷的HLM以填充管。 </li><li>离心机在4℃在130,000 xg下使用SW28水平转头45分钟。允许转子惯性停止（转子减速= 0）。 </li><li>收集漂浮的层，调整到10％的蔗糖和转移到一个13.2毫升超速离心管用于SW41Ti转子，或等效的底部。覆盖密度调节上清液用约5毫升冰冷的100mM碳酸钠缓冲液（pH 11.5）和0.5冰冷HLM毫升。 </li><li>离心机在4℃在274,000 xg下使用SW41Ti水平转头60分钟。允许转子惯性停止（转子减速= 0），以尽量减少脂滴层的破坏。 </li><li>仔细收集含顶部浮动层（ 图2D）脂滴用1毫升吸管，然后重复步骤3.4。 </li><li>离心机在4℃在274,000 xg下使用SW41Ti水平转头30分钟。允许转子惯性停止（转子减速= 0）。 </li><li>仔细收集含脂滴用弯曲巴斯德吸管分为三个1.5毫升管中含有100微升HLM顶白色的浮层。 </li></ol> 表征脂滴组分（协议1和2） 在脂滴分数的回收率和纯度可以通过Western Blot检测结合光或电子显微镜进行验证。此外，可以收集和保存确定净化效率后不同离心步骤等分。在蛋白质印迹法，这是比较合适的，比较脂滴分数表示的全细胞裂解液的当量比在T比较，脂滴至其它膜组分的体积他依据总蛋白含量24。

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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. 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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. 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作者宣称没有竞争的财务权益。

<html> 本协议中的关键步骤 确保培养细胞的生长过程中以与介质和细胞密度是一致的。细胞脂滴是独特的，因为它们相关的蛋白是高度依赖于在其中培养细胞17的环境。因此，其中的细胞生长介质和细胞的密度应密切裂解之前进行监测。 脂滴的蛋白质组合物是酵母细胞的生长阶段的功能。较少的蛋白质将被绑定到液滴在?...

细胞脂滴是具有多种功​​能的细胞活力的细胞器。它们是存储集线器为中性脂质，可被转化成能量，或用于磷脂的合成。液滴发挥中心作用在生理和病理条件，包括动脉粥样硬化，肥胖和相关代谢性疾病，并且还传染病1,2。此外，他们是耐人寻味的来源生物柴油燃料。 对脂滴的细胞作用的许多内容已经从广泛的生物纯化3滴的蛋白质和脂质组分析中得到。这些生物已包括4,5细菌，酵母6-11，12,13植物，线虫14，和苍蝇15,16。由于在人类的代谢性疾病的脂滴的作用的兴趣，液滴也被分离自培养的动物细胞和nimal组织。培养的细胞系已列出的3T3-L1脂肪细胞17，中国仓鼠卵巢（CHO）细胞中的K2 18，人hepatocyes 19,20和上皮细胞系21。动物组织从液滴被分离已包括小鼠骨骼肌22，肝脏23，和乳腺增生病23。如上所述，大多数液滴隔离研究的目的是执行蛋白质组分析的约束因素，并在中性和磷脂脂质组分析。 由于中性脂肪 - 脂滴的最多的部件 - 比大多数其他细胞材料密度较小，传统上一直采用密度梯度离心法进行飞沫隔离。该技术是在这里提出两个的PREPS的核心。以前的技 ​​术6,24相结合，并修改成液滴从培养裂殖酵母细胞的分离的视觉呈现以及从胎盘组织获得的未培养的人类细胞。我们的目标是通过选择两个截然不同的细胞类型作为起始，在液滴分离点，以显示该技术的广泛适用性。这种技术应该是为那些希望以隔离大多数生物滴有用。 方案1描述了从裂殖酵母， 粟酒裂殖酵母 ，它已被用来作为一个模型用于在真核细胞分裂25观察液滴形成脂滴的分离。芽殖酵母酿酒酵母中已被广泛地用来作为模式生物研究脂滴生物学。方案1是适用于两种生物体和在制剂的差异突出显示。 方案2描述了从胎盘绒毛细胞，这是在从人足月胎盘获得的转脂滴的分离。该收集足月胎盘的提供了一个独特的机会，安全和道德得到200-250克现成的人体组织26，其中包含显著数字脂滴的。这是相对于在高等真核生物中，其中液滴从培养的细胞起源最脂滴隔离工作。在这些研究中，脂肪酸，通常加入到培养以促进中性脂质，因而液滴的生长的合成。这是相对于这里的工作，其中脂质小滴在天然条件下在胎盘组织形成。 在脂滴组分的纯度是通过蛋白质印迹分析用细胞器标记的抗体来确定。这两个协议将产生脂滴部分，适合在随后的蛋白质和脂质组分析。

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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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;">Yeast Lytic Enzyme</td>
			<td>MP Biomedicals</td>
			<td style="width:119px;">215352610</td>
			<td></td>
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			<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>
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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;">1 liter glass bottle</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">250 ml flask</td>
			<td></td>
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			<td height="21" style="height:21px;">2.8 liter flasks</td>
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			<td></td>
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			<td height="20" style="height:20px;">2. Yeast lipid droplet isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">PROTOCOL #2&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1. Placental villous cells isolation</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="20" style="height:20px;">Disposable underpads</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">23666062</td>
			<td style="width:167px;"></td>
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			<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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

脂滴是可以在大多数真核细胞和某些原核细胞中找到动态的细胞器。在结构上，所述液滴包括由磷脂单分子层所包围的中性脂质的核心。一种在确定液滴的细胞作用最有用的技术一直是蛋白质组学鉴定结合的蛋白，它可以随着液滴被分离的。在这里，两种方法都描述隔离脂滴和他们结合蛋白从两个广泛的真核生物：裂殖酵母和人类胎盘绒毛细胞。虽然这两种技术有差异，主要方法 - 密度梯度离心 - 是由两个准备共享。这显示了所提出的液滴分离技术的广泛适用性。 在第一协议，酵母细胞是将它们的细胞壁消化酶转化成原生质球。由此产生的原生质球，然后GENTLY溶解在宽松的均质机。聚蔗糖加入到裂解物中，以提供密度梯度，将混合物离心分离3次。第一自旋后，脂质微滴被定位于离心管中的白色的浮动层沿与内质网（ER），质膜，并且空泡。随后的两个自旋是用来消除这些其他三个细胞器。其结果是，仅具有小滴和结合蛋白的层。 在第二协议，胎盘绒毛细胞是从人的足月胎盘酶消化，用胰蛋白酶和DNA酶I。将细胞匀浆在一个宽松的匀浆中分离。低速和中速离心步骤用于去除完整的细胞，细胞碎片，细胞核，线粒体和。蔗糖加入到匀浆，以提供密度梯度，将混合物离心，以脂质小滴从其他cellula分开ř分数。 在这两种协议的脂滴的纯度是通过Western印迹分析来证实。从双方的PREPS液滴部分适用于后续的蛋白质和脂质组分析。

如果密度梯度离心和预期一样，浮层应含有脂滴和在整个的高速旋转的进展耗尽其他细胞器。 对于方案1，西方印迹与标记抗体的脂滴（Erg6p），并且已经发现，与在酵母中，ER（Dpm1p），线粒体（Por1p）脂滴的细胞器相互作用进行的，质膜（Pma1p）和液泡（Vma1p）。 每3自旋的浮动层（步骤3.7，3.10，和3.13）收集等量，沉淀用三氯乙酸（15％终浓度），并溶解...

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

两种技术从1分离细胞的脂滴），酵母细胞和2）人类胎盘介绍。这两个程序的核心是密度梯度离心法，在这里可以用肉眼很容易地可视化含有液滴产生的浮层，提取，并通过Western印迹分析纯度量化。

隔离细胞脂滴：两个纯化技术从酵母细胞与人类的胎盘开始

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

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Bioengineering

19.5K 次观看.  University of Tennessee. 两种技术从1分离细胞的脂滴），酵母细胞和2）人类胎盘介绍。这两个程序的核心是密度梯度离心法，在这里可以用肉眼很容易地可视化含有液滴产生的浮层，提取，并通过Western印迹分析纯度量化。 

视频: 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.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

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

科研

生物工程

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).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

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.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

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.

脂质双层囊泡代使用微流控喷射

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.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

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.

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

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.

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

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.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

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.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

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.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

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