Name: processing of primary brain tumor tissue for stem cell assays and flow sorting
Uploaded: 2012-09-25T11:55:00
Description: Watch this Scientific Journal Video about Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting at JoVE.com

这项工作是由加拿大安大略省癌症研究所（OICR），Terry Fox基金会和美国神经外科医师协会。

1。对脑肿瘤的组织文化<ol><li> 200μL解冻Liberase的（罗氏应用科学部）15毫升的人工脑脊液（ACSF 见表1），并放入37℃水浴。 liberase TM是用于解离主要的组织样本，以及培养tumorspheres的混合的蛋白水解酶。胰蛋白酶-EDTA不同中，Liberase方法保留了表面抗原CD133。对于一个约0.5厘米3的组织样本，我们用200微升Liberase。如果组织是较小的，我们用100微升。 </li><li>带上的氯化铵溶液（?...

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	<li>Ohgaki, H., Kleihues, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+etiology+of+gliomas.">Epidemiology and etiology of gliomas.</a> Acta Neuropathol. 109, 93 (2005).</li><li>Huse, J. T., Holland, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+brain+cancer:+advances+in+the+molecular+pathology+of+malignant+glioma.">Targeting brain cancer: advances in the molecular pathology of malignant glioma.</a> 10, 319 (2010).</li><li>Wechsler-Reya, R., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+biology+of+brain+tumors.">The developmental biology of brain tumors.</a> Annu. Rev. Neurosci. 24, 385 (2001).</li><li>Singh, S. K. <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+a+cancer+stem+cell+in+human+brain+tumors.">Identification of a cancer stem cell in human brain tumors.</a> Cancer Res. 63, 5821 (2003).</li><li>Singh, S. K. <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+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396 (2004).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+and+population+analyses+demonstrate+that+an+EGF-responsive+mammalian+embryonic+CNS+precursor+is+a+stem+cell.">Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell.</a> Dev. Biol. 175, 1 (1996).</li><li>Tropepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+neural+stem+cells+proliferate+in+response+to+EGF+and+FGF+in+the+developing+mouse+telencephalon.">Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon.</a> Dev. Biol. 208, 166 (1999).</li><li>Yin, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AC133,+a+novel+marker+for+human+hematopoietic+stem+and+progenitor+cells.">AC133, a novel marker for human hematopoietic stem and progenitor cells.</a> Blood. 90, 5002 (1997).</li><li>Yu, Y., Flint, A., Dvorin, E. L., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AC133-2,+a+novel+isoform+of+human+AC133+stem+cell+antigen.">AC133-2, a novel isoform of human AC133 stem cell antigen.</a> J. Biol. Chem. 277, 20711 (2002).</li><li>Reya, T., Morrison, S. J., Clarke, M. F., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells,+cancer,+and+cancer+stem+cells.">Stem cells, cancer, and cancer stem cells.</a> Nature. 414, 105 (2001).</li><li>Al-Hajj, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proc. Natl. Acad. Sci. U. S. A. 100, 3983 (2003).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nat. Med. 3, 730 (1997).</li><li>Matsui, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+clonogenic+multiple+myeloma+cells.">Characterization of clonogenic multiple myeloma cells.</a> Blood. 103, 2332 (2004).</li><li>Bao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cancer+stem+cells+through+L1CAM+suppresses+glioma+growth.">Targeting cancer stem cells through L1CAM suppresses glioma growth.</a> Cancer Res. 68, 6043 (2008).</li><li>Bao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioma+stem+cells+promote+radioresistance+by+preferential+activation+of+the+DNA+damage+response.">Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.</a> Nature. 444, 756 (2006).</li><li>Bao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-like+glioma+cells+promote+tumor+angiogenesis+through+vascular+endothelial+growth+factor.">Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.</a> Cancer Res. 66, 7843 (2006).</li><li>Beier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temozolomide+preferentially+depletes+cancer+stem+cells+in+glioblastoma.">Temozolomide preferentially depletes cancer stem cells in glioblastoma.</a> Cancer Res. 68, 5706 (2008).</li><li>Piccirillo, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+pools+of+cancer+stem-like+cells+coexist+within+human+glioblastomas+and+display+different+tumorigenicity+and+independent+genomic+evolution.">Distinct pools of cancer stem-like cells coexist within human glioblastomas and display different tumorigenicity and independent genomic evolution.</a> Oncogene. 28, 1807 (2009).</li><li>Piccirillo, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=morphogenetic+proteins+inhibit+the+tumorigenic+potential+of+human+brain+tumour-initiating+cells.">morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.</a> Nature. 444, 761 (2006).</li><li>Rich, J. N., Bao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+and+cancer+stem+cells.">Chemotherapy and cancer stem cells.</a> Cell Stem Cell. 1, 353 (2007).</li><li>Lapidot, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+initiating+human+acute+myeloid+leukaemia+after+transplantation+into+SCID+mice.">A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.</a> Nature. 367, 645 (1994).</li><li>Reya, T., Morrison, S. J., Clarke, M. F., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells,+cancer,+and+cancer+stem+cells.">Stem cells, cancer, and cancer stem cells.</a> Nature. 414, 105 (2001).</li><li>Fuchs, E., Segre, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+a+new+lease+on+life.">Stem cells: a new lease on life.</a> Cell. 100, 143 (2000).</li><li>Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+units+of+development,+units+of+regeneration,+and+units+in+evolution.">Stem cells: units of development, units of regeneration, and units in evolution.</a> Cell. 100, 157 (2000).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707 (1992).</li><li>Reynolds, B. A., Tetzlaff, W., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multipotent+EGF-responsive+striatal+embryonic+progenitor+cell+produces+neurons+and+astrocytes.">A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes.</a> J. Neurosci. 12, 4565 (1992).</li><li>Hulett, H. R., Bonner, W. A., Barrett, J., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sorting:+automated+separation+of+mammalian+cells+as+a+function+of+intracellular+fluorescence.">Cell sorting: automated separation of mammalian cells as a function of intracellular fluorescence.</a> Science. 166, 747 (1969).</li><li>Kohler, G., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cultures+of+fused+cells+secreting+antibody+of+predefined+specificity.">Continuous cultures of fused cells secreting antibody of predefined specificity.</a> Nature. 256, 495 (1975).</li><li>Barrett, L. E. <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+does+not+predict+tumor+growth+potential+in+mouse+models+of+high-grade+glioma.">Self-renewal does not predict tumor growth potential in mouse models of high-grade glioma.</a> Cancer Cell. 21, 11 (2012).</li><li>Venugopal, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+marks+intermediate+precursors+during+differentiation+of+human+brain+tumor+initiating+cells.">Bmi1 marks intermediate precursors during differentiation of human brain tumor initiating cells.</a> Stem Cell Res. 8, 141 (2012).</li><li>Gerlinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumor+heterogeneity+and+branched+evolution+revealed+by+multiregion+sequencing.">Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.</a> N. Engl. J. Med. 366, 883 (2012).</li><li>Gilbertson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma:+signalling+a+change+in+treatment.">Medulloblastoma: signalling a change in treatment.</a> Lancet. Oncol. 5, 209 (2004).</li><li>Zhu, Y., Parada, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+genetic+basis+of+neurological+tumours.">The molecular and genetic basis of neurological tumours.</a> Nat. Rev. Cancer. 2, 616 (2002).</li><li>Maher, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+glioma:+genetics+and+biology+of+a+grave+matter.">Malignant glioma: genetics and biology of a grave matter.</a> Genes Dev. 15, 1311 (2001).</li><li>Blake, W. J., KAErn, M., Cantor, C. R., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+in+eukaryotic+gene+expression.">Noise in eukaryotic gene expression.</a> Nature. 422, 633 (2003).</li><li>Elowitz, M. B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297, 1183 (2002).</li><li>Maheshri, N., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+with+noisy+genes:+how+cells+function+reliably+with+inherent+variability+in+gene+expression.">Living with noisy genes: how cells function reliably with inherent variability in gene expression.</a> Annu. Rev. Biophys. Biomol. Struct. 36, 413 (2007).</li><li>Raj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+mRNA+synthesis+in+mammalian+cells.">Stochastic mRNA synthesis in mammalian cells.</a> PLoS Biol. 4, e309 (2006).</li><li>Ross, I. L., Browne, C. M., Hume, 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=Transcription+of+individual+genes+in+eukaryotic+cells+occurs+randomly+and+infrequently.">Transcription of individual genes in eukaryotic cells occurs randomly and infrequently.</a> Immunol. Cell Biol. 72, 177 (1994).</li><li>Kubista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Mol. Aspects Med. 27, 95 (2006).</li><li>Nolan, T., Hands, R. E., Bustin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+mRNA+using+real-time+RT-PCR.">Quantification of mRNA using real-time RT-PCR.</a> Nat. Protoc. 1, 1559 (2006).</li></ol>

癌干细胞假说10，是根据工作21在白血病，乳腺癌11和脑癌4,5，表明仅有一个相对小的部分的细胞在肿瘤中，称为肿瘤干细胞，具有客户广泛增殖和自我 - 更新。大多数的肿瘤细胞失去增殖的能力和自我更新，因为它们分化成细胞，成为表型的肿瘤签名。找到关键脑肿瘤的人口能够维持肿瘤细胞的脑肿瘤发生的机制和深入了解，使我们能够追溯起源的细胞。 <p cla...

脑肿瘤中最积极和异质性在人类癌症。虽然他们的早期检测和诊断提供了便利的现代神经影像学技术，我们仍然缺乏许多脑肿瘤的根治性治疗方法，特别是对弥漫性，浸润性，或者那些在大脑深处。 脑肿瘤由于他们的性质非常凶悍，经常无法治愈的儿童癌症死亡率的首要原因。胶质母细胞瘤（GBM），在成人中最常见的原发性脑肿瘤，是一种最积极的人类癌症，担心其均匀致命的预后1。高度恶性星形细胞瘤（WHO 4级），通常发生在成人大脑半球，也可以发生在儿童和婴儿。它的增长是快速渗透，诊断的病理特征包括核多形性，微血管增生，坏死2,3。对于adulTS新诊断为GBM，中位生存期很少超出12个月1，所有治疗方法的普遍反应不佳。我们注意到，有许多共享的成体干细胞和癌细胞的功能和遗传相似性，在肿瘤的分子通路，调节大脑正常发育往往是失调的。在应用的研究脑肿瘤干细胞生物学范式，我们是第一个研究人员前瞻性地识别和纯化的人类的GBMS表现出增殖，自我更新的干细胞特性的细胞亚群，4 和体外分化体内 5。我们最初用来描述正常的神经干细胞（NSCs） 体外 6,7到多个儿童和成人脑瘤的培养条件和检测应用，丰富这些干细胞样细胞的细胞分选的神经祖细胞表面标志CD133 ，9。的CD133 +脑肿瘤馏分包含有高得多的频率的肿瘤起始细胞在NOD-SCID小鼠的大脑5,10比CD133-馏分。这正式成立，只有一种罕见的脑肿瘤细胞与干细胞特性的子集是肿瘤起始，赢得他们的名称为“脑肿瘤启动细胞”或“BTICs”。新的识别BTICs到人脑肿瘤提供了新的见解，给予大力支持的癌症干细胞假说10-13许多实体肿瘤的基础，并建立了一个新的细胞目标的更有效的癌症疗法14-20。重点杀死大量的肿瘤的治疗，可能会错过难得的干细胞样的分数，使肿瘤继续增长。重点杀死癌症干细胞的治疗，可能为脑肿瘤患者提供更好的治疗和预后。 在为了研究BTIC的人群，我们有完善我们的丘尔重新协议，专门选择在人类脑肿瘤的细胞群具有干细胞的特性。无血清，神经干细胞（NS​​C）媒介允许未分化的干细胞状态的维持，和此外，碱性成纤维细胞生长因子（bFGF），表皮生长因子（EGF），白血病抑制因子（LIF），允许多有力，自我更新，和可扩展的人类tumorspheres增殖。在这里，我们描述的原发性脑肿瘤的处理和培养NSC媒体，以丰富BTIC人口参与的方法， 我们都要求我们的实验模型系统“BTIC的的病人分离”强调一个事实，这些都只是最低限度的条件下培养干细胞选择的干细胞群的细胞条件。随后的关键干细胞标志物，如CD133和CD15和流式细胞仪分析免疫标记的BTIC人口中也有描述。然后，我们讨论有限稀释法，这有助于研究自我更新的BTICs潜力。最后，我们将探讨这些稀有细胞的基因表达分析的，分拣单细胞上AmpliGrid幻灯片和执行单细胞RT-PCR。这些技术也适用于其他脑肿瘤如髓母细胞瘤，室管膜瘤和儿科神经胶质瘤。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 1:1的DMEM：F12 </td><td> Invitrogen公司</td><td> 11320-082 </td></tr><tr><td> N2补充</td><td> Invitrogen公司</td><td> 17502-048 </td></tr><tr><td> 1M HEPES </td><td>欧洲野牛</td><td> 330-050-EL </td></tr><tr><td>葡萄糖</td><td> Invitrogen公司</td><td> 15023-021 </td></tr><tr><td> N-乙酰半胱氨酸</td><td> Sigma Aldrich公司</td><td> A9165-25G </td></tr><tr><td>神经的生存因子-1（NSF-1） </td><td>龙沙Clonetics </td><td> CC-4323 </td></tr><tr><td>表皮生长因子（EGF） </td><td> Sigma Aldrich公司</td><td> E9644 </td></tr><tr><td>碱性成纤维细胞生长因子（bFGF） </td><td> Invitrogen公司</td><td> PHG0261 </td></tr><tr><td>白血病抑制因子（LIF） </td><td>密理博</td><td> LIF1010 </td></tr><tr><td>抗生素/霉菌性</td><td>欧洲野牛</td><td> 450-115-EL </td></tr><tr><td> Liberase TM </td><td>罗氏公司</td><td> 05 401 119 001 </td></tr><tr><td>氯化铵溶液</td><td>干细胞技术</td><td> 07850 </td></tr></tbody></table>

processing of primary brain tumor tissue for stem cell assays and flow sorting

脑肿瘤通常是由不同的形态的细胞，表达出各种神经谱系标记。只有小部分的肿瘤细胞与干细胞的特性，称为脑肿瘤启动细胞（BTICs），具备的能力以及多种细胞类型分化，自我更新，并启动体内的肿瘤。我们采用原本用于正常的神经干细胞（NS​​Cs）的各种人脑肿瘤，结果发现，这种文化的方法，具体选择干细胞样的人群的文化条件。无血清培养基（NSC）允许的未分化的干细胞状态的维持，和碱性成纤维细胞生长因子和表皮生长因子的添加允许多强有力的，自我更新，增殖和膨胀tumorspheres。 为了进一步刻画每个肿瘤的BTIC人口，我们通过流式细胞仪检测细胞表面标志物进行评估。我们也可以更具体的characteriz关注的群体排序BULLETIN。自我更新检测到96孔板上执行单BTICs排序条件的形成在37温育后的tumorspheres°C表示的干细胞或祖细胞的存在。某一特定人群的多个细胞的数量也可以在不同的井进行排序，有限稀释分析，分析的自我更新能力。我们也可以研究在一个特定的细胞群的差异表达基因的单细胞RT-PCR。 以下协议描述了我们的程序主人体标本的分离和培养，以丰富的BTIC人口，以及分离的tumorspheres。此外，还包括用于染色，流式细胞仪分析或排序，自我更新的检测，和单细胞RT-PCR方法的协议。

Watch this Scientific Journal Video about Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting at JoVE.com

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

人脑肿瘤发病机制，鉴定的脑瘤开始的细胞（BTICs），罕见的一个异质性肿瘤具有干细胞特性的细胞内，提供了新的见解。我们已调整了特定的文化条件，以丰富BTICs，我们经常使用流式细胞仪检测，以进一步丰富这些人群。对这些孤立的细胞自我更新的检测和RT-PCR单细胞的转录分析可以随后进行。

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Research

JoVE Journal

Medicine

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

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

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 ...

科研

医学

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

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, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...