我们要感谢马塞洛·托罗用流式细胞仪的协助下。这项研究是由瑞典的心脏和肺脏基金会，欧盟委员会第七框架计划“CardioCell”，瑞典研究理事会，亚洲电影大奖保险和ALF的支持。转播是由德意志研究联合会的支持。

1。分离心脏核<ol><li>外套超离心管（贝克曼离心管＃363664）10毫升1％BSA / PBS涂层解决方案。例管，并让他们旋转管旋转30分钟。删除的涂层解决方案，让空气干燥离心管（每管小鼠心脏单小鼠心脏，交替上升至5小鼠心脏或1克的心脏组织的分析，从不同的物种（如人类）可以处理在一管）。 </li><li>以下所有步骤应在冰上进行。解剖用手术刀从新鲜或冷冻单元的小鼠心脏的左心室。请注意，此协议进行了优化，对小鼠心脏，但也可以适应老鼠或人类的心脏。另外，使用1克的心脏组织，从不同的物种。 </li><li>用手术刀修剪成小隔间的标本。 </li><li>组织块转移到一个50毫升的猎鹰裂解液15毫升钢管。 </li><li>均质的心脏组织的T-25超Turrax探头均质（伊嘉）为10秒24,000转。 </li><li>同等体积的裂解液30毫升稀释匀浆。 </li><li>使用玻璃douncer（40毫升）进一步均质的组织和自由的原子核。执行一个大型的间隙杵八杆。 </li><li>通过粗核隔离通过100μm和70μm的尼龙网格过滤器（BD Biosciences公司），连续。 </li><li>原油核自旋向下隔离在一个冷冻离心机（4℃）10分钟在700 XG。 </li><li>小心去除上清相管和擦拭纸巾筒内部。要小心不要打扰细胞核沉淀。 </li><li>溶解粗核蔗糖缓冲5毫升的隔离，吸取解决方案几次上下。添加蔗糖缓冲另有25毫升溶解的颗粒。 </li><li>加入10毫升新鲜制备的蔗糖缓冲涂层的超高速离心管（见第1.1步）。&lt;/ LI&gt; <li>仔细覆盖重悬细胞核从步骤1.9的缓冲蔗糖溶解的颗粒蔗糖缓冲增加了10毫升。 </li><li>之前，他们到JS13.1自由摆动转子平衡离心管中，并放入高速离心机（贝克曼阿凡提的S-25）的转子。 </li><li>在4°C的自旋核样本为60分钟13,000 XG。 </li><li>当自旋已完成，取出管小心从转子和丢弃反相管上清液和擦拭用纸巾管内剩余的碎片。 </li><li>溶解在细胞核存储缓冲区（国安局加上缓冲区）1毫升的核颗粒。注：加国安局包含1.5毫米胺作为一种DNA稳定。 </li><li>进行步骤2.1中，心肌细胞核染色。 </li></ol> 2。免疫组织化学染色的流式细胞仪<ol><li>准备染色阴性对照。以细胞核样品20μL出等分和DD 980μL国安局加缓冲。 </li><li>在1:500稀释到immunolabel心肌细胞核的核样本加入反pericentriolar的材料1抗体（兔抗 - PCM-1，阿特拉斯抗体）。加入相同的稀释步骤2.1准备，作为反PCM-1抗体阴性对照同型抗体。 </li><li>孵育4℃过夜，阴性对照和样品管。 </li><li>阴性对照和样品洗净至少一次与国安局加缓冲（降速管在10分钟的700 XG在冷冻离心机（4℃），弃去上清和细胞核沉淀溶于1毫升国安局加缓冲）。 </li><li>添加抗兔荧光二抗（FITC或APC）在1:1000稀释阴性对照和样品管。 </li><li>在4°C孵育1小时阴性对照和样品管。 </li><li>阴性对照和样品洗净至少一次与国安局，加上缓冲区（700 XG降速管在冷冻离心机（4℃）10分钟。弃上清和细胞核沉淀溶于1毫升国安局加缓冲）。 </li><li>进行流式细胞仪分析和排序。 </li></ol> 3。流式细胞仪<ol><li>大衣核收集管（猎鹰15毫升），1％BSA / PBS溶液，然后再开始流式细胞仪分选步骤1.1中所述。 </li><li>过滤器通过30微米的细胞过滤器的样品和阴性对照和加载第一个阴性对照，流式细胞仪（屋宇署流入）。定义界定汗衫和核（单核），基于前向散射（FSC），前向散射脉冲宽度（FS脉宽）和侧散射（SSC）（ 图2a和b）的第一和第二门。添加样品的DNA染色（DRAQ5（1:500）），可以帮助识别原子核人口的最初。 </li><li>加载immunolabeled样品和定义的第三个门，隔离非心肌细胞的细胞核（PCM-1负）心肌细胞核（PCM-1，收集到阳性）。开始排序（ 图2c和d）。 </li></ol> 可选：为了分析核DNA含量（套数），并进行细胞周期分析，添加适当的DNA染色的原子核（如赫斯特33342或DRAQ5），（ 图2e）。 <ol start="4"><li>经过流式细胞仪分选，放在冰核和重新分析，以确定排序纯度（ 图3a和b）。 </li><li>在15分钟的冷冻离心机在1500 XG分类收集管核自旋向下。 </li><li>细胞核沉淀溶解​​在缓冲区与下游应用兼容。 </li></ol> 4。代表结果细胞核形态和完整性可以通过DNA污渍和评估（ 图1）显微镜观察。评估成功的PCM-1的标签可以通过荧光显微镜和流式细胞仪（ 图1和图2c和d）。 PCM-1-positive和消极的人群应彼此分开（ 图2c和d）。约30％的所有核应该在小鼠左心室心肌细胞核（ 图2d）。排序纯度可通过重新分析排序的细胞核（ 图3a和b）评估。两个原子核的人群应该有一个排序纯度超过95％。 <img alt="图1" src="/files/ftp_upload/4205/4205fig1.jpg" /> 图1。 PCM-1识别心肌细胞核。心肌细胞核（一）与PCM-1（B）和Nkx2.5（三）在成年鼠心脏的抗体染色。 （四）PCM-1标记的细胞核周围心肌细胞的细胞质（肌球蛋白重链（MHC））和心肌细胞核的准确识别，记录PCM-1染色（比例尺为20μm和10表达的转录因子Nkx2.5微米（D，插图））。 （五）心肌细胞核分离可视化与DNA染色DRAQ5。 （f和g）CardiomyocYTE细胞核标记抗体对PCM-1（比例尺为10微米）。请注意，PCM-1 epinuclear染色组织切片的心肌细胞的细胞核和孤立的细胞核（箭头）模式。 <img alt="图2" src="/files/ftp_upload/4205/4205fig2.jpg" /> 图2。流式细胞仪分选 （一） 心肌细胞的细胞核。心肌细胞核确定前向散射（FSC）和侧散射（SSC）。 （二）第二个门标识单核FSC和FS脉冲宽度5。 （C，D）荧光门使心肌细胞核的分离（PCM-1-阳性）和非心肌细胞（PCM-1负），从心脏组织的细胞核。 （五）鼠心肌细胞大多（&gt; 80％）二倍体（2n），只有一小部分是四倍体（4N）6。请注意，人类心肌细胞中含有的多倍体核的频率较高（&gt; 2N）7,8。 <img alt="图3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <stro吴图3。排序的心肌细胞和非心肌细胞的细胞核。重新排序的非心肌细胞（一）和心肌细胞的细胞核（二）分析纯度分析。两个群体排序纯度超过99％。

<ol>
	<li>Ang, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+conventional+approaches+to+identify+myocyte+nuclei+in+histologic+sections+of+the+heart.">Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart.</a> American journal of physiology. Cell physiology. , 298-1603 (2010).</li><li>Bergmann, O. <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+cardiomyocyte+nuclei+and+assessment+of+ploidy+for+the+analysis+of+cell+turnover.">Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.</a> Experimental cell research. 327, 188-194 (2011).</li><li>Bergmann, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Cardiomyocyte+Renewal+in+Humans.">Evidence for Cardiomyocyte Renewal in Humans.</a> Science. 324, 98-102 (1126).</li><li>Walsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+cell+cycle+control+and+growth+estimation.">Cardiomyocyte cell cycle control and growth estimation.</a> Cardiovascular Research. , 1-31 (2010).</li><li>Spalding, K., Bhardwaj, R. D., Buchholz, B., Druid, H., Frisén, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Adler, C. P., Friedburg, H., Herget, G. W., Neuburger, M., Schwalb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+of+cardiomyocyte+DNA+content,+ploidy+level+and+nuclear+number+in+mammalian+hearts.">Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts.</a> Virchows Arch. 429, 159-164 (1996).</li><li>Herget, G. W., Neuburger, M., Plagwitz, R., Adler, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content,+ploidy+level+and+number+of+nuclei+in+the+human+heart+after+myocardial+infarction.">DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction.</a> Cardiovascular Research. 36, 45-51 (1997).</li><li>Adler, C. P., Friedburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+DNA+content.+ploidy+level+and+cell+number+in+geriatric+hearts:+postmortem+examinations+of+human+myocardium+in+old+age.">Myocardial DNA content. ploidy level and cell number in geriatric hearts: postmortem examinations of human myocardium in old age.</a> Mol. Cell Cardiol. 18, 3953-39 (1986).</li><li>Dubois, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRPA+is+a+specific+cell-surface+marker+for+isolating+cardiomyocytes+derived+from+human+pluripotent+stem+cells.">SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.</a> Nature. 29, 1011-1018 (2011).</li><li>Hattori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nongenetic+method+for+purifying+stem+cell-derived+cardiomyocytes.">Nongenetic method for purifying stem cell-derived cardiomyocytes.</a> Nature Methods. 7, 61-66 (2010).</li><li>Fransioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+c-kit-Positive+Cell+Response+to+Pathological+Challenge+in+the+Myocardium.">Evolution of the c-kit-Positive Cell Response to Pathological Challenge in the Myocardium.</a> Stem Cells. 26, 1315-1324 (2008).</li><li>Elliott, 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=NKX2-5(eGFP/w)+hESCs+for+isolation+of+human+cardiac+progenitors+and+cardiomyocytes.">NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.</a> Nature Methods. 8, 1037-1040 (2011).</li><li>Laflamme, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Cardiomyocyte+Repopulation+by+Extracardiac+Progenitors+in+Transplanted+Human+Hearts.">Evidence for Cardiomyocyte Repopulation by Extracardiac Progenitors in Transplanted Human Hearts.</a> Circulation Research. 90, 634-640 (2002).</li><li>Srsen, V., Fant, X., Heald, R., Rabouille, C., Merdes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+proteins+form+an+insoluble+perinuclear+matrix+during+muscle+cell+differentiation.">Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation.</a> BMC cell biology. 10, 28 (2009).</li><li>Spoelgen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometry-based+technique+to+measure+adult+neurogenesis+in+the+brain.">A novel flow cytometry-based technique to measure adult neurogenesis in the brain.</a> Journal of neurochemistry. 119, 165-175 (2011).</li><li>Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+DNA+synthesis+and+binucleation+during+murine+development.">Cardiomyocyte DNA synthesis and binucleation during murine development.</a> The American journal of physiology. 271, H2183-H2189 (1996).</li><li>Olivetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+cardiac+hypertrophy+and+ischemic+cardiomyopathy+do+not+affect+the+proportion+of+mononucleated+and+multinucleated+myocytes+in+the+human+heart.">Aging, cardiac hypertrophy and ischemic cardiomyopathy do not affect the proportion of mononucleated and multinucleated myocytes in the human heart.</a> J Mol Cell Cardiol. 28, 1463-1477 (1996).</li><li>Okada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+sorting+of+neuronal+and+glial+nuclei+from+central+nervous+system+tissue.">Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue.</a> Journal of cellular physiology. 226, 552-558 (2011).</li><li>Matevossian, A., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Nuclei+Isolation+from+Human+Postmortem+Brain+Tissue.">Neuronal Nuclei Isolation from Human Postmortem Brain Tissue.</a> J. Vis. Exp. (20), e914 (2008).</li></ol>

没有利益冲突的声明。

心肌细胞核的准确鉴定为2,3在心肌再生过程的分析是至关重要的。主要是基于传统技术从新鲜组织分离的心肌细胞细胞外基质蛋白消化酶和随后的间质细胞，低速离心净化。从胚胎干细胞（ESC）的生活心肌的进一步净化可进行免疫标记表面标志物，如9或10 SIRPA线粒体染料，固定的心肌细胞可以识别如肌球蛋白重链（MHC）或核蛋白质，如细 ​​胞质标记GATA4或Nkx2.5。然而，N...

<table border="1"><tbody><tr><td> 1。裂解液 </td></tr><tr><td> 试剂名称 </td></tr><tr><td> 0.32米蔗糖</td></tr><tr><td> 10毫米的Tris-HCl（pH值= 8） </td></tr><tr><td> 5毫米氯化钙2 </td></tr><tr><td> 5 mM的醋酸镁</td></tr><tr><td> 2.0毫米EDTA </td></tr><tr><td> 0.5毫米EGTA </td></tr><tr><td> 1 mM的数码地面电视</td></tr></tbody></table><table border="1"><tbody><tr><td> 2。蔗糖缓冲 </td></tr><tr><td> 试剂名称 </td></tr><tr><td> 2.1 M蔗糖</td></tr><tr><td> 10毫米的Tris-HCl（pH值= 8） </td></tr><tr><td> 5 mM的醋酸镁</td></tr><tr><td> 1 mM的数码地面电视</td></tr></tbody></table><table border="1"><tbody><tr><td> 3。原子核存储缓冲区（国安局加上） </td></tr><tR&gt; <td> 试剂名称 </td></tr><tr><td> 0.44中号蔗糖</td></tr><tr><td> 10毫米的Tris-HCl（pH值= 7.2） </td></tr><tr><td> 70毫米氯化钾</td></tr><tr><td> MgCl 2的 10毫米</td></tr><tr><td> 1.5毫米精</td></tr></tbody></table><table border="1"><tbody><tr><td> 试剂和设备 </td><td> 公司 </td></tr><tr><td>同型兔IgG-CHIP级，＃ab37415 </td><td> abcam </td></tr><tr><td>兔抗-PCM-1抗体，＃HPA023374 </td><td>阿特拉斯抗体</td></tr><tr><td>驴秒。抗体，抗兔Alexa的488福陆公司，＃21206或等效秒。荧光抗体</td><td>生命科技</td></tr><tr><td> DRAQ5 </td><td> biostatus </td></tr><tr><td>细胞过滤30微米，70微米和100微米</td><td> BD公司</td></tr><tr><td>玻璃douncer（40毫升）杵“L” </td><td>厂商VWR（惠顿实业公司） </td></tr><tr><td>的T-25的超Turrax均质</td><td>的IKA德国</td></tr><tr><td>分散工具S25的N-18Ğ </td><td>的IKA德国</td></tr><tr><td>贝克曼阿凡提离心机</td><td>贝克曼</td></tr><tr><td>猎鹰管15毫升和50毫升</td><td>厂商VWR </td></tr><tr><td>贝克曼离心管＃363664 </td><td>贝克曼</td></tr><tr><td> JS13.1自由摆动转子</td><td>贝克曼</td></tr><tr><td>涌入流式细胞仪</td><td>贝克曼</td></tr><tr><td>管肩</td><td>厂商VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

鉴定心肌细胞的细胞核已在组织切片挑战，因为大多数的战略仅依靠细胞质标记蛋白1。在罕见的事件，如心肌细胞增殖和凋亡需要的心肌细胞的细胞核的准确识别，分析细胞在病理条件下的稳态和重建。在这里，我们提供了一种方法来隔离验尸组织密度沉淀和免疫标记抗体对pericentriolar材料（PCM-1）和随后的流式细胞仪分选心肌细胞核。这一战略使一个高吞吐量的分析与新鲜组织和冰冻的档案材料，同样的工作的优势和隔离。这使人们有可能已经在生物银行收集研究材料。这项技术是适用和广泛的物种进行测试，适用于多种下游应用，如碳14约会3，细胞CYCLE分析，可视化胸腺嘧啶核苷类似物（如BrdU和IDU）4，转录组和表观遗传分析。

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

心肌细胞核通过密度沉淀分离与1 pericentriolar材料（PCM-1）识别和排序流式细胞仪检测心肌细胞核抗体immunolabeled。

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

isolation-of-cardiomyocyte-nuclei-from-post-mortem-tissue

Research

JoVE Journal

Medicine

16.9K 次观看.  University of Lund. 心肌细胞核通过密度沉淀分离与1 pericentriolar材料（PCM-1）识别和排序流式细胞仪检测心肌细胞核抗体immunolabeled。

视频: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

从部分Pancreatectomized患者的人类胰岛的分离

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

科研

医学

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

光遗传学调节心细胞活性的机电评估

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

使用hiPSC衍生心肌细胞的缺血性心脏病模型和基于视频的心肌细胞收缩比较

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

超声引导诱导多能干细胞来源心肌细胞植入心肌梗死小鼠

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

制备用于心肌梗死后成年小鼠心脏单细胞RNA测序的非心肌细胞悬液

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

使用创新的无异种方法进行人类治疗，分离和培养人脂肪来源的干细胞

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

人瘢痕疙瘩组织中原代真皮成纤维细胞的分离、培养和表征

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...