Name: chromosome preparation from cultured cells
Uploaded: 2014-01-28T13:00:00
Description: Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

在这个手稿中描述的工作成为可能，从帕特里克F.泰勒基金会的资助。

1。贴壁细胞的染色体收获<ol><li> 标准协议 <ol><li>根据特定的细胞培养条件下生长的细胞。当细胞达到对数生长期（80％铺满），加入秋水仙胺的10微升/毫升的细胞培养瓶中。建议最小2×10 6细胞。 </li><li>在5％CO 2培养箱中45分钟，孵育细胞，在37℃。用无菌吸管，从传输介质的细胞成15毫升锥形管。备用。 </li><li>轻轻地加入2ml的HBSS缓冲液加入到烧瓶中洗细胞。漩涡缓?...

<ol>
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我们已经利用本程序为从各种生物体，包括从人获得的各种细胞类型，恒河猴，大鼠和小鼠11,20-22的细胞染色体隔离。标准协议提供的，但某些关键步骤和变量，可能需要调整细胞这些不同的类型。几个具体的步骤是关键的染色体制备和G带，以确保结果的可能的最佳质量两者。一种可影响测定的变量是秋水仙胺孵育时间。时间在秋水仙胺不足会产生更少的中期扩展和更长的，重叠的染色?...

染色体核型分析是利用世界性诊断染色体不稳定和重排，导致遗传性疾病和恶性肿瘤1,2,8,9的常规技术。此外，对于宪法和癌症获得的遗传异常的诊断和研究更高的分辨率可与经典遗传学方法和分子细胞遗传学方法，如荧光原位杂交（FISH）的组合，比较基因组杂交（CGH）来实现和光谱核型分析（SKY）14,15。最近，这些技术已被用于与干细胞研究相关的染色体不稳定性的评价。核型异常如长期培养的胚胎干细胞（ES）和各种生物体的成体干细胞的非整倍体已经报道由多个实验室。最近的证据支持某些细胞系在本质上更倾向于染色体instabiLITY不管文化条件为此，建立和/或维持人，小鼠，或恒河猴干细胞系时，染色体分析，建议作为质量控制过程的一部分。许多报告描述了使用常规和分子细胞遗传学监测干细胞和恶性细胞或有机体的各种文化10-13的染色体的稳定性越来越大的兴趣。这些协议正越 ​​来越多地使用非遗传实验室的培养细胞13染色体的快速评估。我们提出我们的基本程序，用于从不同的细胞类型，这可以应用于从多种生物衍生的临床和研究的目的和细胞染色体制备。

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			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
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			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
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chromosome preparation from cultured cells

染色体（遗传学）分析被广泛用于染色体不稳定性的检测。当随后G显带和如荧光原位杂交（FISH）分子生物学技术，此法必须分析单个细胞对于涉及基因组重排，并涉及一个或多个染色体部分的收益或亏损畸变强大的能力。在人类中，染色体异常发生率约为每1 160个活产1,2，所有流产的60-80％3,4，死胎10％2,5，患有先天性心脏疾病6，3-6％个人13％不孕不育病例2，并在许多患者的发育迟缓和出生缺陷7。恶性肿瘤的细胞遗传学分析是经常使用的研究人员和临床医生，作为克隆性染色体异常的观测已经显示出具有诊断和预后意义8,9。60，染色体分离是非常宝贵的基因治疗和干生物，包括非人灵长类动物和啮齿动物10-13细胞研究。 染色体可以从活组织，包括外周血淋巴细胞，皮肤成纤维细胞，羊膜细胞，胎盘，骨髓和肿瘤样品的细胞中分离出来。染色体在有丝分裂的中期阶段进行分析，当它们被大部分冷凝并因此更加清晰可见。染色体分离技术的第一步涉及主轴纤维的破坏由孵化与秋水仙胺，以防止细胞从进到随后的后期阶段。然后将细胞用低渗溶液，并在其膨胀状态与卡诺固定液保存。的细胞，然后下降到幻灯片，然后可以用于各种各样的程序。 G显带包括胰蛋白酶处理，然后进行染色用姬姆萨打造特色光和暗带。同样的公关ocedure分离的染色体，可用于制备细胞对诸如荧光原位杂交（FISH），比较基因组杂交（CGH），和光谱核型分析（SKY）14,15程序。

高品质的中期息差的染色体分析是必不可少的。一个成功的试验产生的染色体这是很好的传播合适的染色体形态和。正确的G-带染色体包含的特征光与暗的条纹图案。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> 图1：一个成功的染色体蔓延，其中染色体平均长度，以及传播，?...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

染色体可以从活细胞如淋巴细胞或皮肤成纤维细胞中分离出来，并从生物体包括人或小鼠。这些染色体制剂可进一步用于常规G显带和分子细胞遗传学方法，如荧光原位杂交（FISH），比较基因组杂交（CGH）和光谱核型分析（SKY）。

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Research

JoVE Journal

Biology

Counting and Determining the Viability of Cultured Cells

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

科研

生物学

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

果蝇聚乙烯南瓜抗体标记染色体的制备

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

快速风干滴染色体制备方法适用于FISH在植物

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

小鼠精减数分裂染色体的制备

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

缺氧条件下培养细胞内源性核蛋白的共免疫沉淀测定

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...