这项工作是从佛罗里达州的脑瘤研究中心的资助;普雷斯顿A.井小的脑肿瘤治疗中心的支持。

1。收集原发性GBM的组织<ol><li> mutliforme一个胶质母细胞瘤（GBM）肿瘤与癌症的诊断和手术病人获得。 </li><li>神经外科团队必须作出安排。神经外科医生，将放置在一个合适的大小，包含神经干细胞（NS​​C）的培养基辅以10-15％的抗生素（Peniciline / Streptomycine）管切除的大紫荆勋章肿瘤。冷介质，必须提供由实验室手术室。另外，冷的HEPES缓冲的最低限度的基本培养基（HEM）或磷酸盐缓冲生理盐水（PBS）的高浓度抗生素也可以用于此目的。 </li><li>切除的肿瘤组织被传递到冰实验室和置于引擎盖下。 </li></ol> 2。原发肿瘤分解成单细胞悬液<ol><li>原中/ PBS的差额从猎鹰管和样品洗净，用5 - 10M的2-3倍L的PBS /国科会培养基，以清除血液和碎片。 PBS /中等被删除，在培养皿中的大紫荆勋章的肿瘤组织。 </li><li>组织切成小块，剁碎和10号成小片的手术刀刀片胰蛋白酶消化过程，增加表面积。切碎可以采取1-3分钟，取决于肿瘤的大小。 </li><li>碎组织胰酶消化35毫升预热％0.05胰蛋白酶- EDTA在37℃水浴10-15分钟。使用的电子移液器是转移到15毫升猎鹰管的微小肿瘤件和胰蛋白酶。 </li><li>大豆胰蛋白酶抑制剂等体积添加到阻止酶的胰蛋白酶反应后的潜伏期。 </li><li>胰蛋白酶失活，确保移液的悬浮上下几次。然后，暂停800rpm（110克）离心5分钟沉淀下来。 </li><li>上清被丢弃，并组织块悬浮在无菌国科会BA1毫升SAL介质。团块分离轻轻吹打起来，直到达到一个平稳的乳白色单细胞悬液（3-7倍）。步移液操作的数量直接取决于粒子剁碎组织的大小。应避免冗长和充满活力的机械分离，因为它可能导致细胞死亡，并减少在球体形成。 </li><li>要删除联合国分离件和碎片，添加10-15毫升培养基管细胞悬液通过40微米的细胞过滤器过滤后进入一个5​​0毫升管。 </li><li>过滤悬浮离心800rpm（110克）为5min。事后被丢弃上清。 </li><li> Pelletted细胞，然后悬浮在完成国科会为中等细胞计数12毫升。 </li></ol> 3。细胞计数和电镀<ol><li> 10μL的细胞悬液加入到90μL0.04％台盼蓝在1毫升Eppendorf管。 注：其他合适的细胞稀释各条指令可能也可以使用。 </li><li>移液器上下混合暂停。 10μL的细胞/台盼蓝的混合物转移到血球以计数细胞密度。 </li><li>细胞是镀在完成国科会（NSC培养基和NSC增殖的补充，在9:1的比例混合培养基的0.2％肝素（2μg/ml）与20ng/ml EGF，在10ng/ml碱性成纤维细胞生长因子和1μl/ml）适当的组织培养容器。 5，20和40毫升介质的T25，T80和T175培养瓶，分别用于。抗生素可加入浓度为1:100中期，以减少污染的机会。 </li><li>烧瓶放置在一个孵化器设置在37 ° C和5％CO 2。 </li></ol> 4。传代和扩大GBM的衍生领域： <ol><li>当神经球达到了一个直径在150-200微米的平均规模，文化是亚文化的准备。每个烧瓶中的内容被删除，并放置在一个适当大小的无菌TISSUE文化管，并在室温下5分钟800转（110克）离心。 </li><li>除去上清和沉淀重悬于1毫升的0.05％胰蛋白酶EDTA。 </li><li>为了达到最佳的胰蛋白酶消化，细胞悬液培养在37℃水浴为2-3分钟。要停止胰蛋白酶的活性大豆胰蛋白酶抑制剂等体积加入细胞悬液，细胞悬液轻轻吸管上下。 </li><li>细胞悬液，离心5分钟800转（110克）。然后，取上清液被删除，在1毫升的NSC培养基中悬浮细胞。 </li><li>如前面所述，一个细胞计数。 </li><li>这些细胞是镀在完成国科会培养基辅以生长因子，并在适当大小的组织文化，如前面部分所述的船只镀的浓度在5X10 4个细胞/毫升。 </li><li>在7-10天，在37 ° C在潮湿中学神经球形成ified孵化器与5％的CO 2。 </li></ol> 5。代表性的成果： 电镀后从GBM的肿瘤组织中收获了单细胞，肿瘤干细胞样细胞增殖，并产生小的细胞在3-4天，少数细胞组成（见视频和图1）集群。由于这些集群的成长，他们将获得由7-8天这样，适当阶段明亮领域的球形，平均直径为150-200微米的形式（见视频和图2）。在更高的放大倍率，健康的领域，通常体现在其外围microspikes。大球体，如在1-2天内集群文化启动后，由于存在非游离团块文化暴食，不应该被误认为是真正的领域。在原发肿瘤领域的文化碎片量取决于组织的初始源，它是否包含任何周围的脑组织。适当的组织制备技术包括足够数量的中等酶和机械分离，和随后的样品过滤，可以导致在更短的文化碎片。 <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="图1" /> 图1。小学大紫荆勋章后第4天电镀领域的文化。肿瘤干细胞样细胞增殖和细胞产生的小群在3-4天。原放大; 20X。 <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="图2" /> 图2通道电镀后第8天一个大紫荆勋章领域的文化。原放大; 20X。

<ol>
	<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, 1707-1710 (1992).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <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+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods Mol Biol. 750, 61-77 (2011).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <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+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457-e2457 (2011).</li><li>Eramo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15, 504-514 (2008).</li><li>Patrawala, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy.">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl. Cancer. Inst. 100, 672-679 (2008).</li><li>Galli, R. <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+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</li><li>Deleyrolle, L. P. <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+label-retaining+tumour-initiating+cells+in+human+glioblastoma.">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134, 1331-1343 (2011).</li></ol>

没有利益冲突的声明。

为了隔离从正常成人和胎儿大脑1，2，3，4的神经干细胞和祖细胞，也肿瘤干细胞样， 乳腺癌7 6 5肺癌，前列腺癌和脑8，9，如癌组织细胞的神经干检测已经常使用的方法的选择。使用这个简单，重复性实验，可以生成一个无限期的数量从切除的肿瘤组织细胞，表明成体干细胞相似的特点;体内多能，能够创造新的肿瘤后植入，自我更新。这些细胞可用于研究的基本癌的细胞生物学，包括细胞与细胞之间的相互作用，分化，迁移，侵袭和细胞死亡。此外，分离出肿瘤干细胞样细胞提供了一个宝贵的工具，研究如何肿瘤形成，进展和复发，也解开底层产生的细胞机制，最终能提供洞察力，以治疗选项​​。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> NeuroCult NSC基础培养基（人力） </td><td>中等</td><td>干细胞技术</td><td> 05750 </td><td></td></tr><tr><td> NeuroCult NSC增殖补充剂（人力） </td><td>中等补充</td><td>干细胞技术</td><td> 05753 </td><td></td></tr><tr><td> ％0.05胰蛋白酶EDTA </td><td>试剂</td><td> Gibco公司</td><td> 25300-062 </td><td></td></tr><tr><td> *纪念</td><td>试剂</td><td> Gibco公司</td><td> 41500-018 </td><td> HEM的组成部分</td></tr><tr><td> * HEPES </td><td>试剂</td><td>西格玛</td><td> H4034 </td><td> HEM的组成部分</td></tr><tr><td> *蒸馏水</td><td>试剂</td><td&gt; Gibco公司</td><td> 15230-147 </td><td></td></tr><tr><td> ** DNA酶我</td><td>试剂</td><td>罗氏公司</td><td> 104159 </td><td></td></tr><tr><td> **大豆胰蛋白酶抑制剂</td><td>试剂</td><td>西格玛</td><td> T6522 </td><td></td></tr><tr><td> PEN /链球菌</td><td>试剂</td><td> Gibco公司</td><td> 15140-122 </td><td></td></tr><tr><td>第10号手术刀刀片</td><td>手术工具</td><td>屋宇署</td><td> 371610 </td><td></td></tr><tr><td>培养皿</td><td>文化洁具</td><td>屋宇署猎鹰</td><td> 353003 </td><td></td></tr><tr><td>小镊子</td><td>外科手术工具</td><td>精细科学工具</td><td> 11050-10 </td><td></td></tr><tr><td>细胞过滤器</td><td>筛</td><td>屋宇署猎鹰</td><td> 352340 </td><td></td></tr><tr><td> T25烧瓶</td><td>文化洁具</td><td> Nalge怒江NC国际</td><td> 136196 </td><td></td></tr><tr><td> T80烧瓶</td><td>文化洁具</td><td> Nalge NUNC国际</td><td> 178905 </td><td></td></tr><tr><td> 15毫升管</td><td>文化洁具</td><td>屋宇署猎鹰</td><td> 352096 </td><td></td></tr><tr><td> 50毫升管</td><td>文化洁具</td><td>屋宇署猎鹰</td><td> 352070 </td><td></td></tr><tr><td>表皮生长因子</td><td>生长因子</td><td>研发</td><td> 2028 - EG </td><td></td></tr><tr><td> B - FGF </td><td>生长因子</td><td>研发</td><td> 3139 - FB </td><td></td></tr><tr><td>肝素</td><td>生长因子</td><td>西格玛</td><td> H4784 </td><td>重组的PBS </td></tr></tbody></table> *为了使下摆，混合and160ml 1M HEPES的MEM 1x10L包卷到8.75 l使用蒸馏水。设置最终的pH值至7.4ð储存在4 ° C。 **要准备的胰蛋白酶抑制剂解决方案，首先使DNA酶I溶液（100毫克脱氧核糖核酸酶溶解在100毫升下摆）10毫升，再加入0.14克胰蛋白酶抑制剂，DNA酶的解决方案，终于使量高达1升使用HEM。保持在-20 ° C冰箱的最终产品的分装。

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

已被隔离，如乳腺癌，肺癌，结肠癌，前列腺癌和脑肿瘤干细胞样细胞。在所有这些肿瘤的一个关键问题，尤其是在胶质母细胞瘤（GBM）的mutliforme，是识别和分离肿瘤启动细胞的人口（S），以调查他们的作用，在肿瘤的形成，进展和复发。了解肿瘤启动细胞群会提供线索，发现这些肿瘤的有效治疗方法。神经球测定（NSA），由于其简单性和可重复性已被用作隔​​离和传播这种肿瘤细胞的首选方法。此协议表明神经球的培养方法，隔离和扩大人类GBM的手术切除肿瘤组织中的干细胞样细胞。该程序包括一个初始的化学性消化和机械分离肿瘤组织，并随后电镀NSA的文化产生的单细胞悬液。经过7-10天，初级神经球的150-2在直径00微米，可以观察到，并准备进一步传代和扩张。

Watch this Scientific Journal Video about Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay at JoVE.com

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

这个视频协议演示的隔离和扩大干细胞的神经球的实验文化方法如手术切除人脑胶质瘤mutliforme（GBM）的肿瘤组织使用。

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

isolation-expansion-human-glioblastoma-multiforme-tumor-cells-using

Research

JoVE Journal

Neuroscience

29.6K 次观看.  University of Florida. 这个视频协议演示的隔离和扩大干细胞的神经球的实验文化方法如手术切除人脑胶质瘤mutliforme（GBM）的肿瘤组织使用。

视频: Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人力蕈状味觉​​乳头细胞的分离和培养

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

使用F9稀土LACZ基于Cell的报告基因检测在E8.5胚胎和神经球文化相对维甲酸水平的定量测量

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

使用微注射在人赫拉细胞中分体血球定位分析

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

从人包皮中分离、培养和表征原代施旺细胞、角质形成细胞和成纤维细胞

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

人小胶质细胞样细胞：使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...