Ko是支持由美国心脏协会的资助。

在您开始之前成人心<ol><li>刚准备人工血淋巴（AH）的解决方案包含108MM娜5mm的K +，2mm的钙2 +，8MM氯化镁2，1MM的NaH 2 PO 4，4MM碳酸氢钠3，10mm的蔗糖，5mm的海藻糖，5MM的HEPES（pH值7.1，所有的试剂从西格玛化学品）。蔗糖和海藻糖应添加到AH冷藏库存解决方案在使用前，为了防止细菌污染。 </li><li>带来AH室温至少15分钟的含氧空气鼓泡解决方案。 </li><li>拉几个细的毛细血管（如玻璃毛细管，100ul，VWR）去除脂肪。 </li></ol>幼虫的心<ol><li> Sylgard涂层的盖玻片:使用牙签，转移到22x22mm盖玻片约50 -70μlSylgard解决方案。在65℃过夜，让Sylgard变硬。 </li><li>准备Broadie和巴特的缓冲区（135mm的氯化钠，氯化钾5MM，4MM MgCl 2的 ，2MM 氯化钙 ，5mm的工商业污水附加费，36mm的蔗糖，pH值7.15 Broadie和巴特，1993年）。 </li><li>准备申请histoacryl胶所需的工具。首先，拉几个细的毛细血管（例如科学产品GB100T8P）。用小塑料管（聚乙烯，1 / 32"），由较小的一端插入管连接一个1.5毫升枪头。一个具有广泛开放的另一端插入毛细管。 Histoacryl胶的胶水和温和的吸力下降到塑料尖的毛细管浸入负载毛细管。胶工作时，手头应该有第二个玻璃毛细管，以消除过度胶胶水毛细管的尖端。胶水液体，直到它接触到的缓冲液，几秒钟内它就会变硬。但是，它通常仍然是塑料的，足够长的时间从胶水毛细管尖或同时应用到的标本中删除。 </li></ol>从成人飞半完整的果蝇心脏<ol><li>成年果蝇苍蝇飞2-5分钟的小睡（北卡罗来纳州生物供应公司）麻醉。必须小心，不要离开苍蝇在飞午睡长时间曝光。短期受寒也可以用于麻醉苍蝇，但CO 2是不推荐，因为它已不再对心率和节律性的持久的影响。 </li><li>麻醉苍蝇被放在背侧下来，到涂有一层薄薄的凡士林培养皿中。可逆的翅膀和身体的疏水角质层"棒"的果冻，但如果需要的话，可以重新定位的飞。这在随后的操作就显得尤为重要。 </li><li>最初的削减是用弯曲的弹簧剪刀对（Roboz＃5611）。剪刀的刀片放在腿下，向附近的颈部胸部背侧表面角度。单一切割头，腹神经索，腿被删除。然后淹没在一个含氧，人工血淋巴（AH）的解决方案的准备工作。 </li><li>使用弹簧剪刀腹部（包括7和第8腹节）后提示删除与单一的削减。这提供了访问沿着腹部的两个边缘的横向削减和供应切断后肠道和腹部角质层之间的连接。腹腹部角质层中解脱出来的皮瓣，然后删除。 </li><li>在步骤3和4对肠道的前部和后部的连接被切断，使腹部器官，现在只由tracheols地方举行。肠道和其他腹部器官，通常可以作为一个单一的大规模使用珠宝商的镊子（Roboz，＃55）和温柔揪着删除。卸下内部器官，揭示仍然附着在背的角质层是由脂肪组织包围着心脏的跳动管。 </li><li>脂肪组织在光镜下相当不透明的，因此它往往是必要去除一些脂肪，以清楚地看到心管。这是通过使用一个吸脂精心绘制的玻璃毛细管技术。毛细血管是使用标准的吸管拉马（如萨特的P - 97移液器拖轮），然后插入塑料管连接到真空源（聚乙烯，1 / 16"）。这个系统是用来关闭多余的脂肪吸从心管周围。吸力尖端直径成正比，所以不应使用提示直径超过40微米的移液器。必须格外小心避免触及心脏本身，因为后部的心是很脆弱的，该地区应完全避免。 </li><li>现在暴露在成年果蝇心管应跳动。如果有必要，可以重新定位的角质层和附加的心轻轻提起凡士林的角质层，并仔细捣固它背下来，又避免与心脏组织有任何接触。在t他的观点洗澡准备的解决方案，应更换新鲜的AHL。应该允许这种准备与充氧20-30分钟达到平衡之前，任何操作。如果准备工作是要维持的时间超过60分钟，应该灌注新鲜的AHL。 </li></ol>固定化果蝇幼虫光学记录<ol><li> Sylgard盖玻片的每个角落放置一个Histoacryl胶水下降。胶滴加入B＆B的缓冲液50μL，就会变硬即刻。填写的B＆B粘点之间的空间 - 胶水将保持在幻灯片上的缓冲区。 </li><li>流浪L3幼虫放在一块冰浴2分钟的湿纸巾。这将减缓其运动。然后转移到缓冲区和东方背侧（这是缺乏的锯齿皮带的幼虫侧）的幼虫。快速应用一些胶水之间的头部区域和Sylgard。胶水会变硬非常迅速，防止幼虫的头部移动。 </li><li>使用镊子小心抢后气孔动物，轻轻地舒展。沿幼虫双方申请在后半部的胶固定在这个位置上的幼虫。为了减少进一步的运动由体壁肌肉的角质层，动物的侧面添加更多的胶水。幼虫现在是固定在这个位置上。盖玻片放入一个小的Petri菜充满AHL或PBS。心跳，现在可以记录，不受体壁运动的干扰。 </li></ol>代表性的成果: 几个小时半完整的成年果蝇心脏搏动以下清扫时，在保持新鲜，含氧啊（见补充资料，Ocorr，等，2007）。一个代表性的例子是在影片1所示。 第三龄幼虫和年轻的苍蝇（1 - 3周后羽化）心中普遍表现出心脏跳动在定期利率介于1 - 3赫兹。苍蝇比3个星期一般都比较心律失常以上;然而，这些心中仍然能够击败自发地在含氧的AHL小时。在解剖过程的结果损坏的成蝇的心中通常出现极度收缩的局部地区，都不能放松。心跳率超过1 Hz的慢，很少看到苍蝇超过3周的年龄年轻，因此这类制剂被认为是破坏和被丢弃。 <a href="http://www.jove.com/files/ftp_upload/1425/MOVIE1.avi">点击这里下载电影1</a> -一个星期岁成年野生型飞（W 1118实验室应变），显示裸露的腹部心管（前左） 。请注意定期，节律性收缩。在右上角的不透明区域是剩余的腹部脂肪组织;心两侧的圆形细胞是心包的细胞。

<ol>
	<li>Ocorr, K., Reeves, N., Wessells, R. J., Fink, M., Chen, H. -. S. V., Akasaka, T., Yasuda, S., Metzger, J., Giles, W., Posakony, J. W., Bodmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNQ+potassium+channel+mutations+cause+cardiac+arrhythmias+in+Drosophila+that+mimic+the+effects+of+aging.">KCNQ potassium channel mutations cause cardiac arrhythmias in Drosophila that mimic the effects of aging.</a> Proc Natl Acad Sci U S A. 104, 3943-3948 (2007).</li><li>Broadie, K. S., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+embryonic+neuromuscular+synapse+of+Drosophila+melanogaster.">Development of the embryonic neuromuscular synapse of Drosophila melanogaster.</a> J Neurosci. 13, 144-166 (1993).</li></ol>

果蝇模型已被证明是一个强大的遗传工具，已经被用来解决各种科学问题，从胚胎发育，学习和记忆不等。最近这种多功能的模式生物用于研究在果蝇心脏功能的基因。一些试图量化在成年果蝇的心脏生理依赖于完整的苍蝇经腹角质层的意见。这些方法大多依靠肉眼观察或改变录音，通过腹部的量化一个参数，心率在光照强度变送器。这些方法有一些限制:通常只有心前结束，可以观察到，我们正...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Micro Dissecting Spring Scissors (curved)</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5611</td>
<td>Good for gross cuts (Step 3)</td>
</tr>
<tr>
<td>Micro Dissecting Spring Scissors (straight)</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5620</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #55 forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-51</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Glass Capillaries, 100ul</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53432-921</td>
<td>&nbsp;</td>
<tr>
<td>Glass Capillaries, fine</td>
<td>&nbsp;</td>
<td>Science Products</td>
<td>GB100T8P</td>
<td>&nbsp;</td>
<tr>
<td>Pipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Equipment</td>
<td>P-97</td>
<td>Both horizontal and vertical pipette pullers will work</td>
</tr>
<tr>
<td>Plastic tubing</td>
<td>&nbsp;</td>
<td>Tygon R-3603</td>
<td>1/16&#x201D; inner diameter</td>
<td>for 100&mu;l capillaries</td>
</tr>
<tr>
<td>Plastic tubing</td>
<td>&nbsp;</td>
<td>Tygon R-3603</td>
<td>1/32&#x201D; inner diameter</td>
<td>for small capillaries</td>
</tr>
<tr>
<td>Histoacryl&reg; tissue adhesive</td>
<td>&nbsp;</td>
<td>B.Braun, Melsungen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

visualizing the beating heart in drosophila

"<em&gt;果蝇</em&gt;心脏最近出现了一个很好的模型系统研究生肌心中的基本功能的基因，细胞和分子机制。在飞行检查心脏功能的一个关键的一步是寻找一种方法来访问的方式，保留其生肌的功能，同时仍然允许跳动的心脏器官进行观察和记录的心。两种不同的方法，观察和记录中的幼虫和成人心脏跳动<em&gt;果蝇</em&gt;这里描述。成蝇半完整的准备允许在不干扰色素的角质层和覆脂肪体腹部心脏清晰的可视化。记录幼虫心脏跳动，它是必要的，以固定的幼虫，最大限度地减少体壁运动，从而减少心脏运动不与心肌收缩。我们的方法产生稳定的成年和幼虫的心准备，可以在1-3赫兹率击败小时。

Watch this Scientific Journal Video about Visualizing the Beating Heart in Drosophila at JoVE.com

Visualizing the Beating Heart in Drosophila

技术所需的可视化的幼虫和成虫在跳动的心脏<em&gt;果蝇</em&gt;提交。每个人生阶段需要不同的方法。

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

visualizing-the-beating-heart-in-drosophila

Research

JoVE Journal

Biology

Visualizing the Beating Heart in Drosophila

18.6K 次观看.  The Sanford Burnham Institute for Medical Research. 技术所需的可视化的幼虫和成虫在跳动的心脏果蝇提交。每个人生阶段需要不同的方法。

视频: Visualizing the Beating Heart in Drosophila

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

科研

生物学

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...