国家机构的健康，NHLBI（市，PY，PS）。 美国心脏协会（巨浪）。 斯坦福大学，VPUE格兰特（巨浪）。

改编自以前的研究1。 1。 Annexin V的共轭超顺磁性氧化铁<ol><li>氧化0.15 M高碘酸钠（NAIO 4）（4:1重量：重量比）为1小时在黑暗中的解决方案与超顺磁性氧化铁粒子（纳米技术公司，海洋5毫克/毫升）在20°C 2。 </li><li>在20°C孵育氧化12小时的超顺磁性氧化铁，用纯化的的ANX蛋白（比例为1:1，重量：重量）0.15 M四硼酸钠（钠2 B 4 O 7 10H 2） </li><li>进一步减少为1小时与通风柜下硼氢化钠（硼氢化钠4）混合，然后以0.15 M的Tris-HCl解渴。 </li><li> ANX-超顺磁性氧化铁在14000 XG（75微米孔隙单片机过滤器，微孔，比尔里卡，MA）的离心分离免费ANX。 </li><li>进一步完善绑定杂质去除，用磁选设备（海洋，纳米技术公司）的化合物。 </li><li>量化过滤器截留（ANX，超顺磁性氧化铁）蛋白水平Bradford蛋白检测，并存储在-80℃时，2毫克/毫升在0.1 M的Tris-HCl，1％的血清白蛋白和蛋白酶抑制剂（HALT蛋白酶抑制剂，Thermo Scientific的1-50毫克/毫克ANX）。 </li></ol> 2。动态光散射<ol><li>分别稀释在PBS的超顺磁性氧化铁和超顺磁性氧化铁ANX-0.1的光学密度和动态光散射分析1纳米激光粒度仪当值律师服务机（马尔文公司，伍斯特郡，英国）3。 </li><li>获得该化合物为单峰峰值（理想），一个Z-平均粒径和多分散指数（PDI）。与此相比，单独的氧化铁纳米粒子的DLS-测量大小的大小。 ANX-超顺磁性氧化铁DLS结果表明：共轭粒子流体力学直径的78纳米与0.13 1交车，反映了一个同质ANX-超顺磁性氧化铁物种。 </li></ol> 3。细胞文化，文化中的细胞凋亡的诱导，培养细胞的MRI <ol><li>文化成年鼠骨髓间充质小号TEM细胞在培养液（mMSCs的），辅以10％胎牛血清和1％青霉素，链霉素（Invitrogen公司，公司），最好是在流逝，20-30 4。 </li><li>在培养细胞凋亡的诱导治疗不同在37°C，与阿霉素（Sigma公司，1毫米，0.9％的生理盐水，1μM的浓度在媒体）12倍。 </li><li>在培养细胞凋亡的量化使用或者核磁共振与ANX超顺磁性氧化铁（在3.4以下）或使用后，TUNEL染色显微镜（APO-BrdUTM TUNEL法检测试剂盒，分子探针/ Invitrogen公司，尤金，或）或ANX-FITC染色后的金额（瑞士罗氏公司）。 </li><li> ANX-超顺磁性氧化铁（蛋白质10毫克/毫升培养基，除非另有说明），或与同等重量的自由，超顺磁性氧化铁（5毫克）标记的细胞，15分钟，在37°C 1毫升1％琼脂糖，以防止扩散的超顺磁性氧化铁纳米粒子清洗4次，用PBS和覆盖。执行的菜肴MRI（如下所述）。另外，洗细胞和trypsinize后，阿霉素和ANX-超顺磁性氧化铁标记，然后让这些细胞Eppendorf管形成颗粒。 </li></ol> 4， 体外 MRI <ol><li>使用标准的膝盖， 在体外 MRI分析接收线圈1.5或3特斯拉GE公司Signa的EXCITE扫描器。 </li><li>将培养皿中，成琼脂幻象或管和图像使用梯度回波被宠坏的梯度序列：重复时间（TR），回波时间（TE）550毫秒，10毫秒，翻转角15°，矩阵256×256，现场的视图3厘米×3厘米，层厚1.3毫米，平均人数（国家执行）2，间距0毫米5。图片轴向通过与T2 *信号损失地区的利益，一个固定的区域（投资回报）（例如，0.8平方毫米 ）的盘上的细胞层。使用从琼脂幻象控制投资回报率信号调整的背景。测量背景噪声信号从假体周围的空气。 </li><li>计算的MRI对比噪声比（晨晨：] / [Si样品的投资回报率SI控制的投资回报率，其中SI是信号强度，SI的背景噪音的SD]SD是标准差），计算每个培养皿。 </li></ol> 5。代表结果 在体外的T2 *信号损失的测量要求均匀琼脂幻影和缺乏气泡，这将创造信号无效神器的。这件神器是难以区分真正T2的信号损失超顺磁性氧化铁，可解释的结果是至关重要的。空气神器的例子，以及真正的氧化铁与T2 *信号损失，请参阅图1。 在体外培养皿成像，适用于每个培养皿中的琼脂适当的音量，使足够的轴向MRI片，其中约1毫米厚的，可以作出充分的形象整个菜。 图2说明了预期的MRI信号从个人在琼脂培养皿。通常有一些在板的边缘信号的变化，因此INT地区分析erest应该避免这些不平衡的地区。 <img alt="图1" src="/files/ftp_upload/3775/3775fig1.jpg" /> 图1。ANX-超顺磁性氧化铁检测培养的乳鼠心肌细胞凋亡。 图1A显示了颗粒与控制解决方案（左管），ANX，超顺磁性氧化铁（中间管），阿霉素+ ANX-超顺磁性氧化铁（右管）治疗新生儿心肌的照片。注意：棕褐色的阿霉素+ ANX-超顺磁性氧化铁处理的细胞，反映保留在细胞凋亡的ANX超顺磁性氧化铁复合增加。 图1B显示的激烈与T2 *信号损失，阿霉素+ ANX-超顺磁性氧化铁细胞在体外 MRI图像。此外，气泡之间的第 4 次和5 次 Eppendorf管（黑色小信号无效），这是类似的ANX，超顺磁性氧化铁的信号无效，这可能会影响对CNR分析。 <img alt="图2" src="/files/ftp_upload/3775/3775fig2.jpg" /> 图2。ANX-超顺磁性氧化铁约束力是特定文化中的细胞凋亡。 图2A显示凋亡大鼠乳鼠心肌细胞在培养皿中，受到任何ANX FLUOS染色或ANX-超顺磁性氧化铁染色，高，低浓度的ANX，超顺磁性氧化铁（左，右，分别）。请注意不同的T2 *信号损失（黑场信号）荧光ANX（ANX-FLUOS）的水平低与高层次的ANX，超顺磁性氧化铁治疗的细胞凋亡。增加的T2 *信号损失的权利是一个显着减少的ANX-FLUOS信号的，已参加ASX的超顺磁性氧化铁的浓度较高有关。 图2B显示ANX-超顺磁性氧化铁对ANX-FLUOS信号和分离的竞争性结合常数，K I。 X轴表示在日志规模的膜联蛋白的蛋白质浓度。 电影1 <a href="http://www.jove.com/files/ftp_upload/3775/3775movie1.mov" target="_blank">点击这里查看补充</a>电影。

<ol>
	<li>Dash, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+MRI+probe+to+detect+treatment+of+cardiac+apoptosis+in+vivo.">A molecular MRI probe to detect treatment of cardiac apoptosis in vivo.</a> Magn. Reson. Med. , (2011).</li><li>Khym, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+of+methylamine+with+periodate-oxidized+adenosine+5'-phosphate.">The reaction of methylamine with periodate-oxidized adenosine 5'-phosphate.</a> Biochemistry. 2, 344-350 (1963).</li><li>Liu, Y. Y., Yu, Y., Zhang, G. B., Tang, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+controlled+release+of+novel+nanoparticles+based+on+MMA/beta-CD+copolymers.">Preparation, characterization, and controlled release of novel nanoparticles based on MMA/beta-CD copolymers.</a> Macromol. Biosci. 7, 1250-1257 (2007).</li><li>Seo, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FeCo/graphitic-shell+nanocrystals+as+advanced+magnetic-resonance-imaging+and+near-infrared+agents.">FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared agents.</a> Nat. Mater. 5, 971-976 (2006).</li><li>Hsiao, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+death+induced+by+myocardial+ischemia+and+reperfusion:+measurement+with+recombinant+human+annexin-V+in+a+mouse+model.">Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model.</a> Circulation. 102, 1564-1568 (2000).</li><li>Blankenberg, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+and+imaging+of+phosphatidylserine+expression+during+programmed+cell+death.">In vivo detection and imaging of phosphatidylserine expression during programmed cell death.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 6349-6354 (1998).</li><li>Schellenberger, E. 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没有利益冲突的声明。

连接超顺磁性氧化铁Annexin V的共轭方法利用膜联蛋白的侧链胺组和超顺磁性氧化铁纳米粒子的羧基基团。通过特定的氧化还原步骤，这些化合物的共价键可以实现，产生的功能化纳米粒子可以被孤立。此法可推广到其他蛋白质与纳米粒子的利益。 最关键的步骤是氧化还原条件，在适当的温度和温和搅拌。产量低，可能会导致不完全氧化或减少，导致无残留Annexin V的，在过滤?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录＃ </td></tr><tr><td>超顺磁性氧化铁</td><td>海洋纳米技术公司</td><td> ICK-40-005 </td></tr><tr><td>阿霉素</td><td>西格玛</td><td> D1515-10MG </td></tr><tr><td> SuperMag分离</td><td>海洋纳米技术公司</td><td> N / A </td></tr><tr><td>动态光散射纳米激光粒度仪机</td><td>马尔文公司</td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

细胞凋亡是许多疾病的显着特征，这一程序性细胞死亡之前，通常会发生疾病的临床表现是显而易见的。在其最早的，可逆的阶段检测细胞凋亡的一种手段，将承担临床前的&#39;窗口&#39;，预防或治疗措施，在此期间，可以采取保护心脏的永久性损伤。这里我们提出一个简单和可靠的方法，共轭人Annexin V的（ANX），如饥似渴地结合到细胞中最早的，可逆的凋亡阶段，超顺磁性氧化铁（SPIO）纳米粒子，作为检测的MRI造影剂。共轭方法开始的超顺磁性氧化铁纳米粒子，氧化成羧基的超顺磁性氧化铁多糖壳的氧化。纯化蛋白ANX，然后添加一个硼酸钠溶液，以方便与超顺磁性氧化铁在ANX共价相互作用，减少缓冲区的设置。一个与钠borohydrid最后削减步骤e是执行完成减少，然后淬火反应。未结合ANX从离心过滤组合。动态光散射（DLS）的大小和ANX-超顺磁性氧化铁产品纯度验证。这种方法并不需要，或修改，超顺磁性氧化铁壳多糖，反对交联氧化铁粒子共轭方法或生物素标记的纳米粒子。因此，这种方法代表了一个简单，可靠的方法，可以延长至共轭利益的其他蛋白质。

Watch this Scientific Journal Video about Synthesis of an In vivo MRI-detectable Apoptosis Probe at JoVE.com

Synthesis of an In vivo MRI-detectable Apoptosis Probe

细胞凋亡的早期检测可以识别处于危险的细胞群，在多种疾病。在这里，我们展示了一个方法来链接早期凋亡检测蛋白Annexin V的一个MRI检测的氧化铁纳米粒子（超顺磁性氧化铁）。此方法可扩展到其他感兴趣的蛋白质生成MRI检测的分子成像探针。

合成的<em&gt;在体内</em&gt; MRI检测细胞凋亡的探讨

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

synthesis-of-an-in-vivo-mri-detectable-apoptosis-probe

Research

JoVE Journal

Biology

Synthesis of an In vivo MRI-detectable Apoptosis Probe

9.1K 次观看.  Stanford University Medical Center. 细胞凋亡的早期检测可以识别处于危险的细胞群，在多种疾病。在这里，我们展示了一个方法来链接早期凋亡检测蛋白Annexin V的一个MRI检测的氧化铁纳米粒子（超顺磁性氧化铁）。此方法可扩展到其他感兴趣的蛋白质生成MRI检测的分子成像探针。

视频: Synthesis of an In vivo MRI-detectable Apoptosis Probe

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...