KO는 미국 심장 협회에서 교부금에 의해 지원됩니다.

당신이 시작하기 전에 성인 심장 <ol><li> 갓 인공 hemolymph (AH) 108mM 나를 포함하는 솔루션 +, 5mM K +, 2mM 칼슘 2 +, 필름 MgCl 2, 1mM 아니 2 PO 4, 4mm짜리 NaHCO 3, 10mM 자당, 5mM trehalose, 5mM HEPES (산도 7.1의 모든 시약의 준비 시그마 화학). 자당과 trehalose는 세균 오염을 방지하기 위해 사용하는 직전 냉장 주식 솔루션에서 AH에 추가되어 있어야합니다. </li><li> 상온 및 최소한 15 분 공기 버블링에 의해 산소 솔루션 AH 가져와. </li><li> 지방 제거에 필요한 몇 가지 좋은 모세 혈관 (예 : 유리 모세 혈관, 100ul, VWR) 당겨라. </li></ol> 애벌레의 마음 <ol><li> Sylgard - 코팅 coverslips : 22x22mm coverslip에 Sylgard 솔루션의 50 70μl에 대한 이쑤시개, 전송을 사용합니다. ° C 밤이여 65 Sylgard 강화하자. </li><li> Broadie 및 베이트의 버퍼를 (., 산도 7.15 Broadie와 베이트, 1993을 참조하십시오 135mM NaCl, 5mM KCl, 4mm짜리 MgCl 2, 2mM CaCl 2, 5mM TES, 36mM 자당) 준비합니다. </li><li> histoacryl 접착제를 적용하는 데 필요한 도구를 준비합니다. 첫째, 몇 가지 좋은 모세 혈관 (예 : 과학 제품 GB100T8P) 당기십시오. 튜브에 작은 끝부분을 삽입하여 작은 플라스틱 튜브 (Tygon, 1 / 32 &quot;)와 1.5ml 피펫 팁을 연결합니다. 상대방하기 위해 광범위한 개방과 모세를 삽입합니다. 플라스틱 끝에 접착제 부드러운 흡입의 드롭으로 모세관을 찍기로 Histoacryl 접착제와 모세관을로드합니다. 접착제로 작업할 때 하나는 모세 접착제의 일각에서 과도한 접착제를 제거하기 위해 손을 모세관 두 번째 잔을 있어야합니다. 그것은 몇 초 내에됩니다하든 버퍼 호수와 접촉에 와서까지 접착제는 액체이다. 그러나, 그것은 일반적으로 표본에 적용하면서 접착제 모세관 끝부분에서 또는 제거하기 위해 충분히 플라스틱 남아 있습니다. </li></ol> 성인 플라이에서 세미 그대로 Drosophila 헛 <ol><li> 성인 Drosophila의 파리 2~5분에 대한 플라이 냅 (캐롤라이나 생물학 공급 주식 회사)와 anesthetized 있습니다. 관리는 더 이상 노출에 대한 플라이 냅에 파리를 떠날하지 않도록해야합니다. 추위에 단기 노출도 파리를 마취하는 데 사용할 수 있지만 더 이상 심장 박동과 rhythmicity에 대한 지속적인 효과를 가지고 같은 CO 2는 권장하지 않습니다. </li><li> Anesthetized 파리는 바셀린의 얇은 레이어 코팅 페트리 접시에, 지느러미 쪽 아래, 배치됩니다. 날개와 본문에 소수성 표피는 reversibly 젤리에 &quot;스틱&quot;하지만 필요한 경우 비행은 재지정 수 있습니다. 이것은 이후의 조작 중에 특히 중요 해지고 있습니다. </li><li> 초기 컷은 봄 가위 곡선 쌍 (Roboz # 5611)를 사용하여 이루어집니다. 가위 블레이드는 다리 아래에 위치하며 목 근처 흉부의 등의 표면을 향해 아래로 직각입니다. 머리, 복부 신경 코드, 그리고 다리가 싱글 컷과 함께 제거됩니다. 준비 그러면 산소, 인공 hemolymph (AH) 솔루션에 빠져들 수 있습니다. </li><li> 스프링 가위를 사용하여 복부 (복부 세그먼트 7 8 구성된)의 뒷부분 끝은 하나의 절단과 함께 제거됩니다. 이것은 복부의 양쪽 가장자리를 따라 수평 상처를 만들기위한 액세스를 제공하고 사후 창자와 복부 표피 사이의 연결을 끊을 수로 제공하고 있습니다. 복부 복부 표피의 해제 플랩을 뺍니다. </li><li> 복부 장기 이제에만 tracheols의 장소에서 개최하므로 3 단계와 4 단계에서 창자의 앞쪽에 및 사후 연결이 절단됩니다. 직감과 다른 복부 장기는 보통 보석의 포셉 (Roboz, # 55)와 울리는 부드러운을 사용하여 하나의 덩어리로 제거할 수 있습니다. 내장을 제거하면 여전히 지방 기관에 의해 둘러싸인 등의 표피에 부착된 박동 심장 튜브를 보여줍니다. </li><li> 지방 시체는 결과적으로 그것이 명확하게 심장 튜브를 참조하기 위해이 지방의 일부를 제거하는 것이 필요 빛을 현미경에 매우 불투명하고 있습니다. 이것은 잘게 그려진 유리 모세관을 사용하여 지방 흡입 기술에 의해 수행됩니다. 모세 혈관은 표준 피펫 풀러 (예 : 서터 P - 97 피펫 풀러)을 사용하여 만든 다음 진공 소스에 부착된 플라스틱 튜브 (Tygon, 1 / 16 &quot;)에 삽입됩니다. 이 시스템은 심장 관 주위의 여분의 지방에서 흡입하는 데 사용됩니다. 직경 40 마이크론보다 큰 팁을 pipettes를 사용하지 않아야하므로 흡입 힘이 팁 직경에 비례합니다. 심장의 후부 일부 지역은 모두 피해야한다 매우 불안정하기 때문에 익스 트림 치료는 심장 자체를 감동 피하고로 이동해야합니다. </li><li> 성인 Drosophila의 심장 관은 현재 노출되고 치고해야합니다. 필요한 경우 표피와 연결되어 마음이 부드럽게 석유 젤리 밖으로 표피를 리프팅 조심스럽게 다시 내려 다림 다시 심장 조직과의 접촉을 피함으로써 재설정하실 수 있습니다. t에서그의 요점은 준비를 목욕 솔루션은 신선한 AHL로 대체해야합니다. 이 준비는 사전에 어떤 조작으로 산소와 20-30분에 대한 평형 수 있어야합니다. 준비 60 분 이상 지속되는 경우 그것은 신선한 AHL과 perfused해야합니다. </li></ol> 광학 녹음에 대한 Immobilizing Drosophila의 애벌레 <ol><li> Sylgard의 coverslips의 각 모서리에 가까운 Histoacryl 접착제 한 방울을 넣으십시오. 접착제 방울에 B &amp; B 버퍼의 50μl를 추가하는 강화가 즉시됩니다. 접착제는 슬라이드에있는 버퍼를 유지합니다 - B &amp; B와 함께 붙어 장소 사이의 공간을 채우십시오. </li><li> 방랑의 L3 유충을 가지고 2 분 얼음 서부 유럽 표준시 조직 종이에 놓으십시오. 이것은 움직임을 느리게합니다. 그런 다음 버퍼와 방향 등의 측면 접속 (이것은 작은이 벨트 부족 유충의 측면이다)로에 유충을 전송하기만하면됩니다. 신속하게 머리를 지역 및 Sylgard 사이 접착제를 적용합니다. 접착제가 경화는 매우 신속하고 이동에서 유충의 머리를 방지합니다. </li><li> 포셉를 사용하면 조심스럽게 후부 spiracles하여 동물을 잡고 부드럽게 스트레칭. 뒷부분 이분의 일에 유충의 양쪽을 따라 접착제를 적용하여이 위치에 유충을 수정. 동물의 측면에 더 많은 접착제를 추가, 본문 벽의 근육에 의해 표피의 추가 움직임을 줄이기 위해. 유충은 지금이 자리에서 해결되었습니다. coverslip는 AHL 또는 PBS 가득한 작은 페트리 접시에 배치될 수 있습니다. 심장 박동은 지금 몸 벽 움직임의 간섭없이 기록될 수 있습니다. </li></ol> 대표 결과 : 신선한 산소 AH 유지하면 세미 그대로 성인 Drosophila의 마음 (보충 자료를 참조 Ocorr, 외., 2007) 해부 다음 시간 리드미컬 이길 겁니다. 대표 예제는 영화 1 표시됩니다. 제 3 instar의 유충과 젊은 파리 (1 - 3 주 게시 eclosion)에서 마음 - 3 Hz에서 일반적으로 1 사이의 속도로 일반적인 심장 박동을 나타냅니다. 그럼에도 불구하고, 이러한 마음은 여전히​​ 산소 AHL의 시간 동안 자발적으로 이길 수있는, 삼주가 일반적으로 더 arrhythmic보다 나이가 난다. 해부 프로 시저의 결과로 손상 파리 성충의 마음은 일반적으로 휴식을 취할 수없는 극단적인 수축의 지역화된 영역을 보여줍니다. 따라서 이러한 준비가 손상 것으로 간주되어 버립니다, 1 Hz에서보다 느린 심장 박동 률은 거의 나이 삼주 미만 파리에서 관찰되지 않습니다. <a href="http://www.jove.com/files/ftp_upload/1425/MOVIE1.avi">. 영화 1 다운로드하려면 여기를 클릭하십시오</a> 노출된 복부 심장 관을 보여주는 일주 옛 성인 wildtype을 날게 (W 1,118 연구실 변형) (왼쪽 앞쪽에) -. 정규, 리듬 수축을합니다. 오른쪽 상단에 불투명 지역 나머지 복부 지방 기관되며 심장의 양쪽에 둥근 세포 pericardial 세포 수 있습니다.

<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>

Drosophila 모델 embryological 개발에서 학습 및 메모리에 이르기까지 과학 질문 다양한 주소로 사용되었습니다 강력한 유전자 도구로 입증되었습니다. 최근이 다양한 모델 생물이 비행의 심장 기능의 유전자를 조사하는 데 사용되었습니다. 성인 Drosophila의 심장 생리를 정할 시도의 숫자가 복부 표피를 통해 그대로 파리에서 만들어진 관측에 의존합니다. 이러한 방식의 대부분은 하나의 매개 변수, 심?...

<!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; Drosophila</em&gt; 심장이 최근 myogenic 마음에 기​​능을 기본, 유전 세포 및 분자 메커니즘을 조사에 대한 좋은 모델 시스템으로 등장하고 있습니다. 파리의 심장 기능을 검사에서 중요한 단계는 여전히 뛰는 심장 장기 관찰하고 기록할 수 있도록하면서 그 myogenic 기능을 보존하는 방식으로 마음을 액세스하는 방법을 찾는 것입니다. 유충과 성인 모두에서 뛰는 심장을 관찰하고 레코딩을위한 두 가지 방법<em&gt; Drosophila</em&gt; 여기에 설명되어 있습니다. 성인 파리를 사용하여 우리 반 그대로 준비는 색소 상피의 간섭과 지방의 시체를 overlying없이 복부 마음의 명확 시각화 수 있습니다. 애벌레 마음을 녹음하기 위해서는이를 심근 수축과 관련되지 않은 마음의 움직임을 줄여 신체 벽의 움직임을 최소화 유충을 고정하기 위해 필요 없잖아. 우리의 방법은 안정 성인과 1-3 Hz의 속도로 시간 동안 이길 수 애벌레 심장 준비를 생산하고 있습니다.

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

Visualizing the Beating Heart in Drosophila

기술은 애벌레와 어른의 뛰는 심장을 시각화에 필요한<em&gt; Drosophila</em&gt; 제공됩니다. 각 생활 단계는 다른 방법론이 필요합니다.

에 잔 심장을 떠올리 Drosophila</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 조회수.  The Sanford Burnham Institute for Medical Research. 기술은 애벌레와 어른의 뛰는 심장을 시각화에 필요한 Drosophila 제공됩니다. 각 생활 단계는 다른 방법론이 필요합니다.

비디오: 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.

라이브를 떠올리 Drosophila</em형광 염료와 함께> Glial - 신경근육학 융티온

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

연구

생물학

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.

의 형광 라벨링 Drosophila 심장 구조

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.

애벌레의 모니터링 심장​​ 기능 Drosophila melanogaster 생리 연구

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.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

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 &amp;#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.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

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 &amp;#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.

조직 샘플에서 γH2AX Foci의 Quantitation

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 &amp;#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.

이온화 방사선 다음 γH2AX의 공간적 분포 평가

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.

Clonogenic 분석 : 자기편 셀

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.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

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.

칼슘의 평가는 그대로 골격 근육 섬유에서 불꽃

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