Name: isolating and analyzing cells of the pancreas mesenchyme by flow cytometry
Uploaded: 2017-01-28T14:00:00.000+00:00
Duration: 5 min 38 s
Description: Watch this Scientific Journal Video about Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry at JoVE.com

The authors thank Adi Sasson for the technical assistance and Helen Guez for the critical reading of the manuscript. This work was supported by European Research Council starting grant no. 336204.

实验按照委员会关于动物研究在特拉维夫大学批准的方案进行。 1.从小鼠胰腺组织隔离<ol><li> 对于成年小鼠: <ol><li>制备消化缓冲液:0.4毫克/毫升胶原酶P和0.1毫微克/毫升DNA酶I溶解在Hanks&#39;平衡盐溶液（HBSS）中。刚准备5毫升每只小鼠缓冲区的15毫升锥形管，并保持他们在冰上直到使用。 </li><li>根据机构指引安乐死的小鼠。 </li><li>解剖每只小鼠以去除胰腺（为说明，请参见参考文献18）;将其放置在含有HBSS培养皿。有可能在立体显微镜下执行此阶段，并且提高消化效率，切割组织分成2 - 4件。 </li><li>放置胰腺组织成含有消化缓冲液（在步骤1.1.1的方法制备）的管。保持冰管，直到所有的老鼠解剖和胰腺组织集。 </li><li>重复步骤1.1.2 - 1.1.4与剩余的小鼠。 </li></ol></li><li> 对于胚胎和新生小鼠: <ol><li>准备消化缓冲液:0.4毫克/毫升胶原酶P和0.1毫微克/毫升DNA酶I溶解在HBSS。在一个15毫升的锥形管2毫升每只小鼠缓冲 - 在出生后第7天（P7）或更小的胚胎和幼崽，刚准备1.5。在一个15毫升的锥形管4毫升，每鼠缓冲区 - 为P7以上的幼崽，准备3。置于冰上直至使用。 </li><li>根据机构指引安乐死的小鼠。对于胚胎，从母亲的子宫中删除所有的胚胎，并放置在含有PBS 10毫米的培养皿。在一个时间解剖一个胚胎。 </li><li>莱在其后面的鼠标，其头部指向从调查的路程。用细镊子，拉起腹部皮肤和开放的中线暴露腹腔到鼠标隔膜18。 </li><li>用细镊子，分离肝脏FR嗡背面腹壁。随着朝鼠尾连续运动，挖出内脏（包括肝，胃，脾，肠，肾和胰腺），并放置在含有HBSS培养皿。 </li><li>下一个立体显微镜，取出肝脏和肾脏，以暴露胰腺（ 图1）。用细镊子，从胃，十二指肠分离胰腺，最后，从脾。 </li><li>放置胰腺组织成含有消化缓冲液（在步骤1.2.1的方法制备）的管。对于P14以上的幼崽，切割组织成2块，增加消化效率。保持冰管，直到所有的老鼠解剖，并收集胰腺组织。 </li><li>重复步骤1.2.2 - 1.2.6与剩余的小鼠。 </li></ol></li></ol> 2.胰腺消化<ol><li>孵育在加热块含有胰腺组织（在消化缓冲液中）的试管，在37℃与agitat 30分钟离子在700rpm。 15分钟后，手动颠倒他们3撼动管 - 4倍，并把它们放回加热块。 注意:如果使用加热模块，无需搅拌功能，反转管每隔5 - 10分钟。确保组织被适当消化。如果不是这样，切割组织切成小块孵育前或增加搅动。 </li><li>停止组织消化，将管在冰上，加入10毫升冷HBSS的每个管。离心管，在4℃和300×g离心5分钟，吸出上清液。 </li><li> 再悬浮: <ol><li>对成年小鼠和P14或老年人幼仔，重新暂停与6毫升的PBS将沉淀，并通过放置在新的15毫升锥形收集聚丙烯管的顶端70微米的细胞滤网过滤它。以确保最大细胞回收，洗原管与另外的6毫升的PBS，并通过细胞过滤到收集管应变它。 </li><li>对于胚胎和幼崽年轻Ť汉P14，重新悬浮用2毫升的PBS将沉淀，并通过放置在一个5毫升圆底聚苯乙烯收集管（FACS管）的顶部有35微米的细胞滤网过滤它。以确保最大细胞回收，洗原管用另外2毫升的PBS，并通过细胞过滤到收集管应变它。 </li></ol></li><li>离心管，在4℃和300×g离心5分钟，吸出上清液。小心除去液体，如细胞松弛地附着在聚苯乙烯管中。 </li></ol> 3.制备标记单细胞<ol><li>缓冲制剂:对于通过FACS细胞分离，制备混合的PBS（不含氯化钙和氯化镁），5％牛胎儿血清（FBS）和5mM EDTA的排序缓冲器。对于流式细胞仪分析，通过用0.05％的叠氮化钠补充排序缓冲器准备分析缓冲液中。两个缓冲器可以存储在4-8℃下长达2个月。 </li><li>重新暂停排序或ANA细胞裂解液。重新暂停从胚胎和幼仔相比于1mL P7年轻分离各胰脏，从P8和满月在1.5毫升之间幼仔，并且从小鼠中超过一个月老年人在3mL。通过放置在圆底聚苯乙烯试管（FACS管）的顶部有35微米的细胞滤网过滤细胞。 </li><li>用于染色的控制，取出50到从在先前步骤（总样品体积的不超过5％）制备的样品100-μL等分试样，将它们放入新FACS管;包括用于每个荧光管使用，包括DAPI和荧光蛋白，以及用于未染色的对照。 注意:当使用转基因小鼠与表达荧光蛋白的细胞，或当细胞数量是有限的，包括非转基因组织作为染色对照。如果表达荧光蛋白的细胞没有进一步染色使用，请继续执行步骤3.8。 </li><li>降速细胞在300×g离心5分钟，在4℃和吸出上清液。 </li><li>对于集团王，再暂停（辅以山羊IgG 1μL100μL整理或分析缓冲液）与封闭液的细胞孵育在冰上30分钟。 </li><li>染色细胞表面标记物，制备包含在每个样品100微升的体积所有期望的荧光团标记的抗体的混合物。准备在排序或分析缓冲2X抗体稀释液（ 即，稀释抗体，以获得双所需浓度;如果1的最终稀释度:200是期望的，稀释抗体1:100）。未经洗涤封闭溶液，添加100μL的混合物在样品细胞达到200微升的最终染色体积。孵育在冰上30-60分钟，并在黑暗中。 </li><li>到设定的分析或分类参数（参见步骤4.3和5.2），制备仅包含使用（染色对照）抗体的一个额外的管。在分选或分析缓冲液（如在步骤3.6中所述）制备2×抗体稀释液。确保包括一个SI对于使用的每种荧光团，以及未染色对照ngle染色控制稀释。未经洗涤封闭溶液，加入抗体混合物的细胞（在步骤3.3中制备）来实现的200微升最终染色体积100微升。孵育30 - 在冰上并在黑暗中60分钟。 </li><li>填充每个管与分析或分类缓冲器4毫升的最大体积洗。任选地，通过35微米的细胞过滤网重新菌株细胞（如在步骤3.2中所述）。 </li><li>离心在4°C 300 XG 5分钟，小心地取出上清。 </li><li>在分析或分类缓冲器重新悬浮的样品。重悬在500微升的胚胎中分离的细胞，从幼仔相比于1mL1个月年轻化，从1 - 旧3个月2毫升，并从比在3mL 3个月以上的小鼠。染色控制可以被重新悬浮于缓冲液300微升。 </li><li>添加200毫微克/ DAPI毫升到重悬浮的细胞，以确定死亡细胞。确保包括管合作ntaining未染色细胞无DAPI的流式细胞仪的设置。进行到细胞分选（步骤4）或分析（步骤5）。 </li></ol> 4.细胞分选<ol><li> 准备工作: <ol><li>细胞用RNase排序之前RNA提取，涂层的1.5ml收集管抑制剂立即排序之前。为此，加入1毫升排序缓冲器，含有0.01 U / ml的RNA酶抑制剂，以无菌的1.5毫升收集管中。 5分钟后，涡管和除去液体。 </li><li>用于细胞之前细胞培养物分选，添加无菌培养介质的3毫升（Dulbecco氏改良的Eagle培养基（DMEM），补充有20％FBS，1％L-谷氨酰胺和青霉素 - 链霉素1％）到无菌的15毫升收集管。 </li></ol></li><li>装载管进入在FACS分拣机之前，涡旋它简单地重悬浮细胞。置于冰上剩余的管子。 </li><li>通过分析染色的控制，以确定排序参数（ 例如启动， </em&gt;电压和补偿）和分配门（ 例如，总的细胞群体，住DAPI阴性的细胞，细胞群进行排序）。 </li><li>一旦分拣参数和大门都设置了，装载样品并启动细胞分选入收集管。 注:排序条件高度依赖于仪器。我们使用100μm的喷嘴宽度，23.1 psi的压力，和5的最大排序速度。 </li><li>继续RNA提取或排序细胞的培养。 注:对于RNA提取，离心细胞以2000×g离心5分钟，并用一个标准提取协议继续之前除去多余的液体。用于培养细胞，如果细胞在非无菌条件下进行排序，由填充培养用培养基的管中并以尽量减少其污染培养前在300 xg离心离心它为7分钟洗两次。 </li></ol> 5.通过流式细胞仪细胞分析<ol><li> l在oading每个管进入细胞仪，涡流它简单地重悬浮细胞。置于冰上剩余的管子。 </li><li>通过以确定分析参数（ 例如，电压和补偿）分析未染色和单染色样品开始。 </li><li>一旦分析参数被设置，加载每个样品，包括染色控制，并记录结果。分析使用流式细胞分析软件所获得的结果。 </li></ol>

<ol>
	<li>Gu, G., Dubauskaite, J., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+the+pancreatic+lineage:+NGN3++cells+are+islet+progenitors+and+are+distinct+from+duct+progenitors.">Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.</a> Development. 129 (10), 2447-2457 (2002).</li><li>Cabrera, O., Berman, D. M., Kenyon, N. S., Ricordi, C., Berggren, P. -. O., Caicedo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unique+cytoarchitecture+of+human+pancreatic+islets+has+implications+for+islet+cell+function.">The unique cytoarchitecture of human pancreatic islets has implications for islet cell function.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (7), 2334-2339 (2006).</li><li>Gittes, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology+of+the+pancreas:+a+comprehensive+review.">Developmental biology of the pancreas: a comprehensive review.</a> Developmental biology. 326 (1), 4-35 (2009).</li><li>Villasenor, A., Cleaver, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+the+developing+pancreas+and+its+blood+vessels:+An+evolving+dialog.">Crosstalk between the developing pancreas and its blood vessels: An evolving dialog.</a> Seminars in cell &amp; developmental biology. , 1-8 (2012).</li><li>Landsman, L., Nijagal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+mesenchyme+regulates+epithelial+organogenesis+throughout+development.">Pancreatic mesenchyme regulates epithelial organogenesis throughout development.</a> PLoS Biology. 9 (9), e1001143 (2011).</li><li>Lammert, E., Cleaver, O., Melton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pancreatic+differentiation+by+signals+from+blood+vessels.">Induction of pancreatic differentiation by signals from blood vessels.</a> Science. 294 (5542), 564-567 (2001).</li><li>Pierreux, C. E., Cordi, 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=Epithelial:+Endothelial+cross-talk+regulates+exocrine+differentiation+in+developing+pancreas.">Epithelial: Endothelial cross-talk regulates exocrine differentiation in developing pancreas.</a> Developmental biology. 347 (1), 216-227 (2010).</li><li>Golosow, N., Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitheliomesenchymal+interaction+in+pancreatic+morphogenesis.">Epitheliomesenchymal interaction in pancreatic morphogenesis.</a> Developmental biology. 4, 242-255 (1962).</li><li>Bhushan, A., Itoh, 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=Fgf10+is+essential+for+maintaining+the+proliferative+capacity+of+epithelial+progenitor+cells+during+early+pancreatic+organogenesis.">Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis.</a> Development. 128 (24), 5109-5117 (2001).</li><li>Larsen, B. M., Hrycaj, S. M., Newman, M., Li, Y., Wellik, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Hox6+function+is+required+for+mouse+pancreatic+endocrine+cell+differentiation.">Mesenchymal Hox6 function is required for mouse pancreatic endocrine cell differentiation.</a> Development. 142 (22), 3859-3868 (2015).</li><li>Sasson, A., Rachi, E., 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=Islet+pericytes+are+required+for+beta-cell+maturity.">Islet pericytes are required for beta-cell maturity.</a> Diabetes. 65 (10), 3008-3014 (2016).</li><li>Stanley, S. A., Kelly, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+electromagnetic+control+of+the+hypothalamus+regulates+feeding+and+metabolism.">Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism.</a> Nature. 531 (7596), 647-650 (2016).</li><li>Brissova, M., Aamodt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+microenvironment,+modulated+by+vascular+endothelial+growth+factor-A+signaling,+promotes+&#946;+cell+regeneration.">Islet microenvironment, modulated by vascular endothelial growth factor-A signaling, promotes &#946; cell regeneration.</a> Cell metabolism. 19 (3), 498-511 (2014).</li><li>Nikolova, G., Jabs, 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=The+vascular+basement+membrane:+a+niche+for+insulin+gene+expression+and+Beta+cell+proliferation.">The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation.</a> Developmental cell. 10 (3), 397-405 (2006).</li><li>Rhim, A. D., Oberstein, P. E., 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=Stromal+elements+act+to+restrain,+rather+than+support,+pancreatic+ductal+adenocarcinoma.">Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma.</a> Cancer cell. 25 (6), 735-747 (2014).</li><li>&#214;zdemir, B. C., Pentcheva-Hoang, T., 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=Depletion+of+carcinoma-associated+fibroblasts+and+fibrosis+induces+immunosuppression+and+accelerates+pancreas+cancer+with+reduced+survival.">Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival.</a> Cancer cell. 25 (6), 719-734 (2014).</li><li>Russ, H. A., Landsman, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Proteomic+Analysis+of+Pancreatic+Mesenchyme+Reveals+Novel+Factors+That+Enhance+Human+Embryonic+Stem+Cell+to+Pancreatic+Cell+Differentiation.">Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.</a> Stem cells international. 2016, 6183562 (2016).</li><li>Gout, J., Pommier, R. M., 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=Isolation+and+Culture+of+Mouse+Primary+Pancreatic+Acinar+Cells.">Isolation and Culture of Mouse Primary Pancreatic Acinar Cells.</a> Journal of Visualized Experiments. (78), e50514 (2013).</li><li>Verzi, M. P., Stanfel, M. 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=Role+of+the+homeodomain+transcription+factor+Bapx1+in+mouse+distal+stomach+development.">Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development.</a> Gastroenterology. 136 (5), 1701-1710 (2009).</li><li>Tribioli, C., Frasch, M., Lufkin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bapx1:+an+evolutionary+conserved+homologue+of+the+Drosophila+bagpipe+homeobox+gene+is+expressed+in+splanchnic+mesoderm+and+the+embryonic+skeleton.">Bapx1: an evolutionary conserved homologue of the Drosophila bagpipe homeobox gene is expressed in splanchnic mesoderm and the embryonic skeleton.</a> Mech Dev. 65 (1-2), 145-162 (1997).</li><li>Hecksher-Sorensen, J., Watson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+splanchnic+mesodermal+plate+directs+spleen+and+pancreatic+laterality,+and+is+regulated+by+Bapx1/Nkx3.2.">The splanchnic mesodermal plate directs spleen and pancreatic laterality, and is regulated by Bapx1/Nkx3.2.</a> Development. 131 (19), 4665-4675 (2004).</li><li>Walmsley, G. G., Rinkevich, Y., Hu, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Fibroblast+Harvest+Reveals+Surface+Marker+Shift+In+Vitro.">Live Fibroblast Harvest Reveals Surface Marker Shift In Vitro.</a> Tissue Engineering Part C: Methods. , (2014).</li><li>Morris, J. P., Greer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicer+regulates+differentiation+and+viability+during+mouse+pancreatic+cancer+initiation.">Dicer regulates differentiation and viability during mouse pancreatic cancer initiation.</a> PloS one. 9 (5), 95486 (2014).</li><li>Miyatsuka, T., Matsuoka, T. -. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronological+analysis+with+fluorescent+timer+reveals+unique+features+of+newly+generated+&#946;-cells.">Chronological analysis with fluorescent timer reveals unique features of newly generated &#946;-cells.</a> Diabetes. 63 (10), 3388-3393 (2014).</li><li>Sneddon, J. B., Borowiak, M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+embryonic-stem-cell-derived+progenitors+by+organ-matched+mesenchyme.">Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme.</a> Nature. 491 (7426), 765-768 (2012).</li><li>Guo, T., Landsman, L., Li, N., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+expressed+by+murine+embryonic+pancreatic+mesenchyme+enhance+generation+of+insulin-producing+cells+from+hESCs.">Factors expressed by murine embryonic pancreatic mesenchyme enhance generation of insulin-producing cells from hESCs.</a> Diabetes. 62 (5), 1581-1592 (2013).</li><li>Olive, K. P., Jacobetz, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Hedgehog+signaling+enhances+delivery+of+chemotherapy+in+a+mouse+model+of+pancreatic+cancer.">Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.</a> Science. 324 (5933), 1457-1461 (2009).</li><li>Raz, Y., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inflammatory+vicious+cycle:+Fibroblasts+and+immune+cell+recruitment+in+cancer.">An inflammatory vicious cycle: Fibroblasts and immune cell recruitment in cancer.</a> Experimental cell research. 319 (11), 1596-1603 (2013).</li></ol>

The authors have nothing to disclose.

在这里，我们描述了分离和分析胰腺微环境细胞的方法。这种方法可用于间充质细胞从胚胎和成年胰腺组织隔离。此外，我们成功地使用该协议来隔离从成人和新生儿胰腺5,17内皮细胞。然而，这可能不适合用于获得胰腺上皮细胞的可重复的单细胞悬浮液（可替代的协议在参考文献18中，23中描述，和24）。使用这种方法，荧光标记的细胞中，无论是表达荧光蛋白或免疫染色表面标记，可以通过FACS进行纯化或通过流式细胞仪分析。 RNA可以从纯化细胞提取介绍他们的基因表达模式。可选择地，纯化的细胞可以培养以建立用于随后的蛋白组学分析的细胞系。这种方法将使因素e的表征胰腺微环境，从而规范其器官，生理和病理生理学xpressed。 胰腺间叶细胞通过促进前体和分化的细胞5,9增殖支持组织器官。这些细胞被示出为支持人类胚胎干细胞的膨胀（人类胚胎干细胞）衍生的胰腺祖细胞17，25，26。因此，描绘的胚胎间充质因素的身份将促进目前的努力，以产生由hESCs和诱导多能干细胞（iPS细胞），为潜在的治疗糖尿病产生胰岛素的β细胞。小鼠遗传学研究允许的生长因子，如FGF10，由间质中产生的识别期间胰腺发育的早期阶段，以促进胰腺上皮膨胀3,9。与识别所述胚胎间充质表达的附加因素的目的，我们分离使用激光捕获显微切割这些细胞中，提取它们的RNA，并进行基因表达分析26。然而，除了是劳动密集的，这种方法依赖于识别基于其形态特征，其中上皮的分支进入周围间质（ 即，E12.5）之前限制了它的使用，以发育阶段的细胞。在后来的发展阶段的特征的间质细胞，我们采用这里5,17中记载的方法。 我们使用这种方法新生儿胰腺间质5，分析表面标志物的表达。此外，间充质细胞从Nkx3.2 -Cre的胚胎和新生儿胰组织中分离; R26-EYFP小鼠，是根据它们的荧光标记在这个鼠标线，并进行培养，建立细胞系17。这些细胞允许由胰腺间质与促进人类胚胎干细胞衍生的胰腺祖17的能力分泌因子识别的蛋白质组学分析。我们进一步用这种细胞的分离方法，以从RNA提取和基因表达分析17成人胰腺组织纯化的间充质细胞。因此，这种方法可以用于识别由胰腺间充质表达的基因和蛋白质，具有支持胰腺细胞发育的能力。 胰腺间充质细胞进一步显示出在胰腺肿瘤发生的作用。 PDAC的特征在于形成由成纤维细胞，免疫细胞的丰富成纤维结缔组织增生基质，和ECM 27。虽然基质被认为推动了许多发展类型的癌症中，显示抑制PDAC进展15，16，28。这表明，胰基质组分分泌抑制肿瘤发生的因素。此外，在间质细胞的组合物，以及在细胞表型的变化可以背后上的上皮细胞15，16，28的作用。因此这里描述的方法可以协助在表征不同类型的细胞，使一个PDAC基质相比，健康胰腺组织。这将进一步允许不同基质的细胞类型的纯化PDAC进展过程中表征其基因表达谱电位变化。然而，由于肿瘤27期间在胰腺癌的ECM组合物的变化，组织消化参数的调整，如纳入ADDIT的有理胶原酶类型或增加孵育时间，可能是需要的。

能量平衡和哺乳动物食物的消化依赖于适当的胰腺功能。成人胰腺由两个主要细胞区室的:外分泌和内分泌。外分泌细胞，包括产生和分泌消化酶和运输这些酶肠道管细胞，包括总胰1质量的80％以上的腺泡细胞。内分泌细胞，包括生产胰岛素的β细胞和胰高血糖素生产的α细胞中，在该嵌入外分泌组织和分泌的激素来调节血糖水平2胰岛被组织。 胰腺细胞通过一个高度调节，多步骤的过程3获得其分化命运。有证据表明，由神经元细胞，内皮细胞，和间质细胞提供外在线索引导在叔胰腺细胞的分化和增殖他胚胎3，4，5。一个例子是主动脉早期胰前体6的规范的要求。后来在发展，被示出的内皮细胞中都胰腺内分泌和外分泌细胞的发展中发挥中心作用，并促进β细胞分化4，6，7。被示出的间质细胞，以支持存活和共同胰祖细胞的扩增，主要是通过生长因子FGF10 8,9的分泌。我们已进一步显示，这些细胞支持内分泌和外分泌的前体在胚胎胰腺5的扩散，以及分化的细胞（包括腺泡和β细胞）。最近，间充质细胞还证明是调节内分泌细胞的分化10。 在成人中，β细胞功能和质量均表现依赖于细胞中的微环境，包括神经元细胞，免疫细胞和内皮细胞，以及周细胞11，12，13。在损伤，均表现内皮细胞招募免疫细胞到胰腺，促进β细胞复制13。内皮细胞进一步示出，以产生细胞外基质（ECM）组件以支持胰岛素表达和β细胞功能14。我们最近表现出胰岛周细胞对β细胞功能11的要求。最后，被示出的胰基质的细胞来调节胰腺导管腺癌（PDAC）15，16的进展。然而，标识指导胰腺发育，功能和肿瘤外在线索的实体，在很大程度上是未知。 识别由胰腺微环境的细胞提供线索需要表征由这些细胞表达的基因和蛋白质。这依赖于以和/或建立的细胞系进行基因表达和蛋白质组分析中分离从胰腺这些细胞。这里，我们提出了一种方法，通过利用表达荧光蛋白或免疫荧光标记的细胞或细胞组织的酶消化和荧光激活细胞分选（FACS）对胰腺微环境的间充质细胞分离。成功地执行该协议，以分离和分析黄色荧光蛋白（YFP）-expressing胚胎，新生儿和成人胰腺5，17间充质细胞。

<table><tbody><tr><td>Collagenase P</td><td>Roche</td><td>11213865001</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-15KU</td><td>The effective units should be at least 2,000 unitz/mL protein</td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt solution (HBSS)</td><td>Sigma-Aldrich</td><td>H6648</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biological Industries</td><td>04-127-1A</td><td></td></tr><tr><td>EDTA</td><td>Biological Industries</td><td>01-862-1A</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Goat IgG serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>RNAse inhibitor</td><td>Invitrogen</td><td>N8080119</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>11965092</td><td></td></tr><tr><td>L-Glutamine</td><td>Biological Industries</td><td>03-020-1B</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Biological Industries</td><td>03-031-1B</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td>Without Calcium Chloride and Magnesium Chloride</td></tr><tr><td>1.5 mL tubes</td><td>Sarstedt</td><td>72 690</td><td></td></tr><tr><td>15 mL conical tube</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>Round Bottom Polystyrene 5 mL tube</td><td>Corning</td><td>352008</td><td>FACS tube. Make sure tube is compatible with the flow cytomter to be used, as there are slight differences in required tubes between brands</td></tr><tr><td>5 mL tube with 35 &amp;mu;m cell strainer Snap Cap</td><td>Corning</td><td>352235</td><td>FACS tube</td></tr><tr><td>70 &amp;mu;m cell strainer&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-098-462</td><td></td></tr><tr><td>Heating block with agitation&amp;nbsp;</td><td>Eppendorf</td><td>ThermoMixer C</td><td></td></tr><tr><td>Centrifuge</td><td>ThermoFisher</td><td>Heraeus Megafuge 40R</td><td></td></tr><tr><td>Steromicroscope</td><td>Nikon</td><td>SMZ 745</td><td></td></tr><tr><td>Cell sorter</td><td>BD Biosciences</td><td>FACSAria IIu</td><td></td></tr><tr><td>flow cytometer</td><td>Beckman Coulter</td><td>Gallios&amp;nbsp;</td><td></td></tr></tbody></table>

isolating and analyzing cells of the pancreas mesenchyme by flow cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

胰腺间质的发展和成年期间需要。这里所描述的方法允许间质细胞从胚胎，新生儿和成人胰腺的隔离。间充质细胞，但没有其他类型的细胞，表达Nkx3.2 -Cre的胰腺黄色荧光蛋白（YFP）; R26R -YFP小鼠5，11，17，19。在开发期间，Nkx3.2（也称为BapX1）由胚胎胰腺，胃和肠间质，以及在骨架体节19，20，21的一个子集表示。该基因在胰腺间充质中表达从E9.5至E11.5，允许来自E9.5 5 Nkx3.2 -Cre的控制下的基因表达， </suP&gt; 19，20。基于此标记，细胞可从大容量的胰腺组织用流式细胞术纯化。 图2显示了从胚胎，新生儿和成人胰腺组织，分离为这里所描述的单细胞的流式细胞仪分析。而与非转基因胰腺组织不含有荧光细胞，Nkx3.2 -Cre; R26R -YFP从所有分析的年龄胰腺组织含有不同的YFP标记的细胞群体（ 图2;标有门）。 下面这里描述的方法中，表达荧光蛋白的细胞可以是纯化的或通过流式细胞仪分析，有或没有附加的免疫染色。例如，根据荧光标记排序后，间质细胞可以培养建立的细胞系（至少5代）， 如图3A所示 。注ŧ<html>他fibrocytic培养的细胞，典型到间充质细胞的形态。此外，使用该系统由分选的细胞来分析基因表达。为此，RNA从排序的间充质细胞中提取合成cDNA和基因的表达水平通过qPCR分析。这样的分析表明，有序细胞表达泛间充质标记物波形蛋白（ 图3B）。最后，由胰腺细胞表面标记物表达可通过流式细胞术进行分析。例如，我们分离从Nkx3.2 -Cre的胰腺组织细胞; R26R -YFP使用此处描述和与细胞表面糖蛋白CD9，将其报告给由成纤维细胞22表示染色它们的方法成年小鼠。如在图3C中所示，所有的荧光标记的Nkx3.2 -Cre细胞; R26R -YFP胰腺表达CD9。 tp_upload / 55344 / 55344fig1.jpg"/&gt; 图1:胚胎和新生儿的胰腺组织。隔离整个胃肠道，包括胃，脾，肠，胰腺，一个E15.5胚胎（A）和P4小狗（B）的。胰腺组织划定了蓝线。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55344/55344fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55344/55344fig2.jpg" /> 图2:间质细胞荧光标记的Nkx3.2 -Cre; R26R -YFP胰腺组织。从非转基因（左图）和Nkx3.2 -Cre分离胰腺细胞的流式细胞仪数据; R26R -YFP在不同年龄的转基因（右图）小鼠:胚胎（A），新生儿（B），和广告ULT（C）。分析细胞的侧向散射（y轴）和黄色荧光（x轴）。盖茨（标有"D"）指示在转基因小鼠中的YFP +细胞群的存在，但不是在非转基因对照。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55344/55344fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55344/55344fig3.jpg" /> 图3:孤立的胰腺细胞分析。从Nkx3.2 -Cre的胰腺组织排序（A）细胞; R26R -YFP（如在图2B中所述）新生小鼠中培养，建立的细胞系。培养的细胞进行成像荧光（绿色;右和左小图）和相位对比（灰色;右图）。 （B）显示Vimentin1（Vim1）棒图表达水平通过YFP + -sorted细胞Nkx3.2 -Cre的胰腺组织; R26R -YFP成年小鼠（如在图1C中描述的;绿色）相比，未分选的胰腺组织（黑）。提取RNA，并通过定量PCR分析基因表达;表达标准化为环素 。 N = 4，*** P &lt;0.001。数据代表平均值±SD。 （C）从Nkx3.2 -Cre分散胰腺细胞的流式细胞仪数据; R26R -YFP成年小鼠免疫染色别藻蓝蛋白（APC）缀合的抗CD9抗体和分析的APC（y轴）和黄色荧光（x轴）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55344/55344fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry at JoVE.com

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

这里，我们描述了一种用于细胞在胰腺微环境从胚胎，新生儿和成年小鼠组织的隔离，着眼于间充质细胞的分离。此方法允许细胞基因表达和蛋白质分泌的分析以阐明调节胰腺发育，功能，和肿瘤发生的外在信号。

隔离并通过流式细胞仪分析胰腺间充质细胞

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

isolating-analyzing-cells-pancreas-mesenchyme-flow

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

12.1K 次观看.  Sackler Faculty of Medicine, Tel Aviv University. 这里，我们描述了一种用于细胞在胰腺微环境从胚胎，新生儿和成年小鼠组织的隔离，着眼于间充质细胞的分离。此方法允许细胞基因表达和蛋白质分泌的分析以阐明调节胰腺发育，功能，和肿瘤发生的外在信号。 

视频: Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

科研

癌症研究

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

发育生物学

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

制备髓源性抑制细胞从幼稚和胰腺癌荷瘤小鼠，用流式细胞仪和自动磁性细胞分选（AutoMACS）（MDSC）

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

医学

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

胰腺组织解剖以分离活的单细胞

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

生物学

从小鼠肢体肌肉中分离卫星细胞和流式细胞术分析

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

作者聚焦:小鼠肌肉干细胞中的 Cistrome 分析 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

一种用于FACS分离的小鼠卫星细胞的CUT&amp;RUN分析的有效方案

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

小鼠肠间充质分离导致端细胞高产量

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

隔离并通过流式细胞仪分析胰腺间充质细胞

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此视频中的章节

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

制备髓源性抑制细胞从幼稚和胰腺癌荷瘤小鼠，用流式细胞仪和自动磁性细胞分选（AutoMACS）（MDSC）

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

胰腺组织解剖以分离活的单细胞

人胰腺癌移植瘤的干细胞的分离

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

小鼠肠间充质分离导致端细胞高产量