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。漩涡缓冲，然后用移液管除去。丢弃。 </li><li>加入1 ml的胰蛋白酶，确保它涵盖了烧瓶的整个表面上。唯一留下的细胞在胰蛋白酶进行约2分钟。一旦大部分细胞已经分离，吸取在锥形管中的介质倒流到细胞上。 </li><li>转移的细胞悬浮液在10毫升的等分试样分成15毫升锥形管中。离心机在200×g离心10分钟。去除上清，重悬沉淀。 </li><li>加入10毫升0.075M的氯化钾已预热至37℃到在锥形管中剩余的粒料。涡流管在中速混合KCl和细胞。 </li><li>培养细胞在37℃，10分钟。离心机在200×g离心5分钟，在25℃下除去上清液（直到约0.5ml遗体），重悬沉淀。 </li><li>小心地加入5毫升新鲜卡诺固定液（3:1比率的甲醇:冰醋酸）的细胞，同时涡旋。然后加入5 ml更多固定液没有涡旋的总体积为10ml。 </li><li>离心机在200×g离心5分钟。去除上清，重悬细胞。加入5 ml固定液到每个管中。 </li><li>离心机在200×g离心5分钟。去除上清，重悬细胞。加入5 ml固定液到每个管中。细胞现在可以存储在4℃可保存一年。 </li></ol></li><li> 修改协议:染色体harvesti淋巴母细胞系的NG <ol><li>根据特定的细胞培养条件下生长的细胞。 （使用烧瓶适当的培养细胞的量）。当细胞达到对数生长期（约两天后的细胞分裂），加入秋水仙胺的10微升/毫升的细胞培养瓶中。 </li></ol></li><li> 修改协议:染色体从全血中收获 植物血凝素（PHA），从红菜豆衍生的外源凝集素，是一种强有力的促细胞分裂剂对人T细胞16。加入PHA的在培养后72小时，约45％的细胞处于S期。这代表了高峰期有丝分裂活性，并且是最佳点时收获的染色体研究。其它有丝分裂原如美洲商陆（1-10微克/毫升）在分析B细胞17,18可被使用。 <ol><li>收集至少有1毫升全血的绿色顶端肝素钠管。使用内采集后3天。血液储存在室温未直到准备使用。 </li><li>等分部分0.25的全血在10毫升含L-谷氨酰胺（20％胎牛血清，1％青霉素/链霉素，1％两性霉素B，和1％PHA）的完全RPMI介质中的溶液。培养在37℃，5％CO 2。 </li></ol></li><li> 修改协议:染色体从骨髓采集 对骨髓细胞遗传学分析是有用的在许多恶性血液疾病为观察染色体异常克隆的可能诊断为白血病的特定类型。染色体的研究也用于评估疾病的进展，如急和对治疗的反应的发生。自骨髓细胞活跃分裂的，不分裂原刺激是必要9,19。对于急性白血病24和48小时的文化也成立。 <ol><li>收集至少1毫升骨髓的绿色顶端肝素钠管。等分0.25骨髓在10ml含L-谷氨酰胺（2完全RPMI介质中的溶液0％胎牛血清，1％青霉素/链霉素，1％两性霉素B）。培养在37℃，5％CO 2。 </li></ol></li></ol> 2。玻片制备和染色固一位代表幻灯片的快速评估将继续进一步的程序，如G显带，FISH，CGH，或Sky之前提供关于收获的质量信息。一些实验室倾向于固体染色的细胞进行快速采集和染色体评估。可替代地，在相差显微镜可用于这种分析。幻灯片最好准备当湿度约为50％，而环境温度（20-25℃）。 <ol><li>离心细胞，在200×g离心5分钟，在25℃下除去上清液，直到只有0.3-0.5毫升遗体。 </li><li>经过沉淀，吸液管3滴从约2中的距离的细胞悬浮液轻轻重悬在其上倾斜约45°的角度，并允许该悬浮液的滑动横跨滑动与滚动。添加一个大的一滴新鲜卡诺固定液的幻灯片。 </li><li>干滑的背部在纸巾上，然后坐在滑板，取出，晾干至少10分钟。幻灯片应完全干燥。 </li><li>准备新鲜的姬姆萨染色溶液（3:1比例格尔缓冲区和姬姆萨染色的）。将载玻片上染色架。覆盖在姬姆萨染色液整个幻灯片。让载玻片保持在5分钟的染色溶液。幻灯片冲洗用蒸馏水，排水，并让​​其自然风干。 </li><li>加入4滴Permount和盖玻片的幻灯片。确保有盖玻片下无气泡。该excess Permount可以与纸巾除去。 </li><li>根据10倍和100倍的放大倍率光学显微镜分析细胞。如果中期细胞丰富，以及扩散，剩下的幻灯片可以被用于其它实验程序。 </li></ol> 3。 G显带用胰蛋白酶和姬姆萨（GTG） &lt;P&gt;胰蛋白酶是在变性DNA区域常染色质组蛋白具有较高的转录活性产生的蛋白水解酶。下面的姬姆萨染色，这些地区将出现光带。高度浓缩的染色质，很少或没有转录活性（异）将其从组蛋白的胰蛋白酶保护的很大一部分，因此，会弄脏黑暗以下​​Giemsa染色。至关重要的是最初G带一张幻灯片监控条件并在必要时调整胰岛素的时间为随后的幻灯片。 <ol><li>添加下面的解决方案，以4科普林氏罐子。 缸＃1 = 30毫升的1X HBSS和4ml的10×胰蛋白酶（0.5％） 罐＃2 = 50毫升的1X的HBSS 缸＃3 = 45毫升的1X HBSS和5毫升胎牛血清的 罐＃4 = 50毫升的1X的HBSS </li><li>沉浸在罐＃1每张幻灯片持续5秒，快速冲洗罐中＃2，互留在滑罐＃3至少30秒，快速冲洗罐中＃4，然后让片干燥。 </li><li>准备FRESħ姬姆萨染色溶液（3:1比例格尔缓冲区和姬姆萨染色的）。将载玻片上染色架。覆盖在姬姆萨染色液整个幻灯片。让载玻片保持在5分钟的染色溶液。 </li><li>在蒸馏水中清洗玻片以相同的顺序，他们进行染色。使载玻片干燥约10分钟。幻灯片应完全干燥。 </li><li>加入4滴Permount和盖玻片的幻灯片。确保有盖玻片下无气泡。过量Permount可以用纸巾除去。 </li><li>根据10倍和100倍的放大倍率光学显微镜分析细胞。调整基于所述第一滑动件的结果的幻灯片的其余部分的胰蛋白酶定时。 </li></ol>

<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。

<table border="0" cellpadding="0" cellspacing="0" width="662">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<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>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

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，很?...

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 ...

chromosome-preparation-from-cultured-cells

Research

JoVE Journal

Biology

80.7K 次观看.  Louisiana State University Health Science Center. 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）。 

视频: Chromosome Preparation From Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

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

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

个别细胞散为主细胞学标本的细胞块的制备

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes 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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

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 activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

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

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

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

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

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

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

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

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 the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei 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 ...