Name: phagocytosis assay for apoptotic cells in drosophila embryos
Uploaded: 2017-08-03T14:00:00.000+00:00
Duration: 10 min 6 s
Description: Watch this Scientific Journal Video about Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos at JoVE.com

感谢Kaz Nagaosa和Akiko Shiratsuchi的意见。

准备<ol><li> 新鲜葡萄汁琼脂平板的制备 <ol><li>加入100毫升水至4.4克琼脂，加热微波炉中的混合物溶解琼脂。 </li><li>向琼脂溶液中加入80 mL新鲜葡萄汁，5 mL乙酸和5 mL乙醇。 </li><li>用移液管将约1.5 mL的溶液倒入每个载玻片上，使其固化。 </li></ol></li><li> 6厘米培养皿琼脂糖板的制备 <ol><li>向0.5g琼脂糖中加入50mL水，并在微波炉中加热混合物以溶解琼脂糖。 </li><li>将大约2.5 mL琼脂糖溶液倒入带有移液管的6 cm盘中。 </li></ol></li></ol>第16期胚胎收集<ol><li>收集成年苍蝇，并将200只雌性和200只雄性添加到50mL锥形管中。 </li><li>将一盘新鲜的葡萄汁琼脂放在酵母中放入50 mL管中，并用海绵盖上页。 </li><li>在16°C的温度下孵育苍蝇2 - 3天。每天更换一次板。 </li><li>在25°C将苍蝇移至黑暗1 h。 </li><li>将旧板更换为新的，不含酵母。让苍蝇放鸡2小时。 </li><li>收集板并在16℃孵育26小时。 </li><li>用油漆刷将板从板上收集到含有0.2％（v / v）Triton X-100的1mL PBS中。 </li><li>用含有0.2％（v / v）Triton X-100的PBS洗涤胚胎两次。 </li><li>为了去除绒毛膜，向胚胎中加入1.2mL次氯酸钠溶液（2.2-3.4％Cl）3分钟。 </li><li>用含有0.2％（v / v）Triton X-100的PBS洗涤胚胎4次。 </li><li>将胚胎放置在6厘米盘的琼脂糖板上。在罗伯茨20所述的显微镜下拿起16号胚胎。用微量移液管在1.5 mL的Treff微量试管中收集大约50个胚胎。 </li></ol>准备胚胎细胞配给<ol><li>用150μLPBS清洗胚胎两次。 </li><li>在PBS中用200μL胶原酶（0.25％（w / v））存在下，在1.5mL微量试管和沉淀混合器中使胚胎均质30次。 </li><li>通过移液10次分散胚胎细胞，并将细胞悬液移至1.5mL管中。在37℃下在水浴中孵育细胞1分钟。重复此步骤两次。 </li><li>在1400×g下添加PBS的800μL细胞悬浮液，和离心机在4℃下5分钟。 </li><li>取出上清液，加入PBS中的200μL胰蛋白酶（0.25％（w / v））至细胞。通过移液分离胚胎细胞50次。 </li><li>用70μm细胞过滤器过滤细胞悬浮液。 </li><li>为了停止胰蛋白酶活性，向过滤的细胞悬浮液中加入40μL热灭活的胎牛血清。加入800μLPBS，4℃下以1400× g离心5分钟。 </li><li>去除超新星个nt，暂停沉淀的细胞在PBS中的200μL，和离心机在1400×g下在4℃下5分钟。 </li><li>去除上清液，并将沉淀的细胞悬浮在30μL的PBS中。 </li><li>将制备的细胞悬浮液装载在氨基丙基三乙氧基硅烷涂覆的玻璃载片上，并等待10 - 15分钟，直到细胞附着在载玻片上。 </li><li>用移液器移除玻璃载玻片上的剩余溶液。将60-70μL含有4％（w / v）PFA（多聚甲醛）的PBS悬浮在细胞上10-15分钟进行固定。 </li><li>取出固定溶液，并将玻璃载玻片放入PBS中超过1分钟。 </li></ol> 4.血细胞染色<ol><li> 用抗Croquemort抗体进行免疫染色 <ol><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片在甲醇中连续浸泡10分钟，然后在PBS中10分钟。 </li><li>在PBS中装载20μL的5％（v / v）全猪血清ng 0.2％（v / v）Triton X-100在细胞上进行阻断。在室温下孵育玻片20分钟。 </li><li>移除玻璃载片上的溶液中，并在含有0.2％PBS安装抗Croquemort抗血清19，21（0.1％（V / V））的20μL（V / V）的Triton X-100细胞上。在4℃下孵育载玻片过夜。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片浸泡10分钟5次。 </li><li>在PBS中浸泡玻片10分钟。 </li><li>在室内的细胞上装载含有0.2％（v / v）Triton X-100和5％（v / v）全猪血清的PBS中的20μL碱性磷酸酶标记的抗大鼠IgG（0.5％（v / v）温度1 h。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片浸泡10分钟5次。 </li><li>将含有100mM Tris-HCl，pH9.5,100mM NaCl和50mM MgCl 2的缓冲液中的载玻片浸泡10分钟。 </li><li>含有0.23mg / mL 5-溴-4的载体磷酸酶底物溶液（BCIP），0.35mg / mL硝基蓝四唑（NBT），100mM Tris-HCl，pH9.9,100mM NaCl和50mM MgCl 2 。 </li><li>在显微镜下观察细胞是否正常染色。当血细胞颗粒中出现强烈的紫色信号时，取出底物溶液，并将载玻片浸泡在由10mM Tris-HCl，pH8.5和1mM EDTA组成的缓冲液中。建议染色约5 - 10分钟。 </li></ol></li><li> 用抗GFP抗体进行免疫染色（血细胞染色的另一种选择） <ol><li>将载玻片在甲醇中连续浸泡10分钟，含有0.2％（v / v）Triton X-100的PBS 10分钟，PBS 10分钟。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的5％（v / v）全猪血清装入20μL用于阻断的细胞上。在室温下孵育玻片20分钟。 </li><li>取出载玻片上的溶液，安装20μL小鼠抗GFP抗体（1％（v / v））/ PBS c在细胞上培养0.2％（v / v）Triton X-100。在室温下孵育玻片一夜。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片浸泡10分钟5次。 </li><li>在PBS中浸泡玻片10分钟。 </li><li>在室内的细胞上装载含有0.2％（v / v）Triton X-100和5％（v / v）全猪血清的PBS中的20μL碱性磷酸酶标记的抗小鼠IgG（0.5％（v / v）温度1 h。 </li><li>将含有0.2％（v / v）Triton X-100的PBS中的载玻片浸泡10分钟5次。 </li><li>将载玻片在100mM Tris-HCl，pH9.9,100mM NaCl和50mM MgCl 2组成的缓冲液中浸泡10分钟。 </li><li>将含有0.23mg / mL BCIP，0.35mg / mL NBT，100mM Tris-HCl，pH9.9,100mM NaCl和50mM MgCl 2的磷酸酶底物溶液装载在细胞上。 </li><li>在显微镜下观察细胞是否正常染色。当紫色信号出现在全血细胞中时，取出底物溶液，并将缓冲液中的载玻片浸泡10mM Tris-HCl，pH8.5和1mM EDTA。建议染色约10 - 15分钟。 </li></ol></li></ol> 5. TUNEL染色细胞凋亡细胞<ol><li>将PBS在PBS中浸泡5分钟。 </li><li>用新的PBS重复。 </li><li>将平衡缓冲液（见材料表）放在细胞上10分钟。 </li><li>从玻片上取出溶液，并在37℃下将20μL含有5μL末端脱氧核苷酸转移酶的TdT溶液和15μL反应缓冲液装入细胞1小时。 </li><li>从玻片上取出溶液，并将缓冲液浸入缓冲液中，缓冲液由0.5 mL STOP / Wash Buffer和17 mL水组成。 </li><li>在PBS中浸泡载玻片5分钟3次。 </li><li>在室温下将20μL抗地高辛 - 过氧化物酶载于细胞上30分钟。 </li><li>将载玻片在PBS中浸泡5分钟4次。 </li><li>将载玻片浸泡在30mL含有pH7.5的50mM Tris-HCl的过氧化物酶底物溶液中3,9&#39;-二氨基联苯胺四氯化氢（DAB）和0.002％（v / v）H 2 O 2的全微微铲30秒。 </li><li>重复步骤5.9直到凋亡细胞染成棕色。将水玻片浸泡在水中以停止过氧化物酶反应。 </li><li>用水封闭细胞观察。 </li></ol> 6.测量凋亡细胞的吞噬水平<ol><li>观察样品在光学显微镜下，以评估吞噬的水平。计数Croquemort阳性细胞或GFP阳性细胞作为血细胞，TUNEL阳性细胞作为凋亡细胞，双重阳性细胞作为吞噬血细胞。 </li><li>吞噬指数定义为双重阳性细胞数与Croquemort阳性细胞或GFP阳性细胞总数的数量。建议观察超过300个血细胞。 </li></ol>

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作者宣称他们没有竞争的经济利益。

<html>我们在此描述了使用果蝇胚胎的吞噬作用。通过使用分散的胚胎细胞定量测量吞噬作用，将血细胞， 果蝇专业吞噬细胞免疫染色用于血细胞标记Croquemort或srpHemo-驱动的GFP，并且在该方案中通过TUNEL检测凋亡细胞。吞噬水平通过计数血细胞总数和吞噬血细胞来表达为吞噬指数。使用分散的胚胎细胞使我们能够观察全胚胎中的所有吞噬细胞和凋亡细胞，并精确量化吞噬水平。?...

在后生动物， 如 线虫 ，果蝇果蝇和小鼠和人类，大量细胞的发育过程中发生凋亡来塑造自己的身体，并在成年后保持动态平衡1，2。凋亡细胞需要快速去除，因为它们通过释放免疫原性的细胞内物质而在周围组织中引起炎症3 。为了便于快速去除，本凋亡细胞所谓吃我信号由吞噬细胞的吞噬受体识别，并通过吞噬作用3，4，5，6被消除。因此，吞噬作用在维持宿主体内平衡方面起关键作用，因此阐明了分子<html>凋亡细胞吞噬的潜在机制是重要的。 负责凋亡细胞的吞噬功能的机制似乎在线虫，果蝇和小鼠7种之中进化上保守的。目前可以使用几种吞噬作用来评估这些模型动物中凋亡细胞的吞噬。在秀丽隐杆线虫中 ，131个体细胞在发育期间经历程序性细胞死亡，细胞尸体被非专业吞噬细胞的相邻细胞吞噬8 。因此，计数线虫中剩余细胞尸体的数量表示体内吞噬水平。通过搜索线虫突变体，显示死细胞数量的增加，吞噬作用所需的几个基因已被鉴定和基因表征9，10，S ="外部参照"&gt; 11,12。 原代培养吞噬细胞（通常是巨噬细胞）的离体吞噬作用常常用于小鼠。使用细胞系如Jurkat细胞制备凋亡细胞，并与原代吞噬细胞混合。孵育数小时后，计数吞噬细胞和吞噬吞噬细胞的总数，以评估吞噬水平。作为这种方法的一个复杂的变形例中，永田的小组开发的体外吞噬作用测定与细胞中表达一个抗胱天蛋白酶ICAD（抑制剂胱天蛋白酶活化的DNA酶），其中凋亡细胞不进行细胞凋亡的DNA片段化，但DNA仍裂解时细胞被吞噬。当这些细胞在吞噬作用测定中用作凋亡靶点时，只有吞噬凋亡细胞的DNA被破碎，并通过TdT介导的dUTP缺口末端标记（TUNEL）染色。所以，水稻通过在吞噬细胞和凋亡细胞的混合物中计数TUNEL信号来测量凋亡细胞吞噬13 。 在果蝇 ，名为血细胞，巨噬细胞果蝇 ，专业的吞噬细胞负责细胞凋亡14，15的吞噬作用。除了与培养的细胞系在体外吞噬作用测定法， 体内用全果蝇胚胎的吞噬作用测定法是可用的。 果蝇胚胎是用于检查凋亡细胞吞噬的水平，因为许多细胞发生凋亡和胚胎发育14，15，16时由血细胞吞噬的有力工具。 体内吞噬试验的一个实例是法国集团开发的方法。在他们的方法中，血细胞是de通过免疫染色过氧化物酶，血细胞标志物，凋亡细胞用核染料，7-氨基放线菌素D在整个果蝇胚胎中染色，双阳性细胞数被计为吞噬信号17 。对胚胎的吞噬作用测定的另一个例子是基于上述Nagata方法的概念;然而， 在体内的吞噬作用是使用DCAD的胚胎进行评估（ 果蝇胱天蛋白酶活化的DNA酶）突变体蝇18,19。这些体内吞噬试验可用于原位直接观察吞噬作用。然而，难以排除计数吞噬细胞步骤中的任何可能的偏差，因为由于其厚度难以观察全胚胎中的所有吞噬细胞和凋亡细胞。 为了克服这个限制，我们开发了在果蝇胚胎中进行新的吞噬作用测定。在我们的方法中，为了容易地计数吞噬血细胞，将整个胚胎均匀化以制备分散的胚胎细胞。通过吞噬细胞标志物的免疫染色检测吞噬细胞，并用这些分散的胚胎细胞用TUNEL检测凋亡细胞。使用分散的胚胎细胞使我们能够测量体内吞噬作用水平，好像我们进行了精确量化吞噬水平的体外吞噬作用。如果它们发展到胚胎20的第16阶段，则吞噬作用的凋亡细胞清除率最丰富的发育阶段，则可以在该测定中使用所有基因型的苍蝇。该方法具有定量评估吞噬水平的优点，因此有助于鉴定参与体内凋亡细胞吞噬的新分子。

<table><tbody><tr><td>whole swine serum</td><td>MP Biomedicals</td><td>55993</td><td>For bloking</td></tr><tr><td>Treff micro test tube(easy fit)&amp;nbsp; Dnase, Rnase free tube, 1.5 mL</td><td>TreffLab</td><td>96. 4625. 9. 01</td><td>For&amp;nbsp; homogenization</td></tr><tr><td>pellet mixer&amp;nbsp; 1.5 mL</td><td>TreffLab</td><td>96. 7339. 9. 03</td><td>For&amp;nbsp; homogenization</td></tr><tr><td>Collagenase</td><td>Sigma-Aldrich</td><td>C-0130</td><td>For preparation of embryonic cells</td></tr><tr><td>Trypsin</td><td>Thermo Fisher SCIENTIFIC</td><td>27250-018</td><td>For preparation of embryonic cells</td></tr><tr><td>Kpl Anti-Rat IgG (H+L) Ab MSA, AP</td><td>KPL</td><td>475-1612</td><td>secondary antibody for&lt;br/&gt; stainig hemocytes with an anti-Croquemort antibody</td></tr><tr><td>5-bromo-4-chloro-&lt;br/&gt; 3-indolyl-phosphate</td><td>Roche</td><td>11383221001</td><td>BCIP, For staining of hemocytes</td></tr><tr><td>nitro blue tetrazolium</td><td>Roche</td><td>11383213001</td><td>NBT, For staining of hemocytes</td></tr><tr><td>Anti-Croquemort antibody</td><td></td><td></td><td>described previously in Manaka &lt;em&gt;et al.&lt;/em&gt;, J. Biol. Chem., 279, 48466-48476</td></tr><tr><td>&amp;nbsp;Anti-GFP&lt;br/&gt; from mouse IgG1&amp;kappa; (clones 7.1 and 13.1)&amp;nbsp;</td><td>Roche</td><td>11814460001</td><td>For staining of hemocytes</td></tr><tr><td>Goat Anti-Mouse IgG-AP Conjugate</td><td>Bio-Rad</td><td>170-6520</td><td>secondary antibody for stainig hemocytes with an anti-Croquemort antibody</td></tr><tr><td>Apop Tag Peroxidase In Situ Apoptosis Detection Kit</td><td>Millipore</td><td>S7100</td><td>For staining of apoptoitc cells. This kit includes Equilibration buffer, Reaction buffer, STOP/Wash buffer, TdT enzyme, and Anti-Digoxigenin-Peroxidase.</td></tr><tr><td>3,3&#039;-diaminobenzidine&lt;br/&gt; tetrahydrichloride</td><td>nacalai tesque</td><td>11009-41</td><td>DAB, For staining of apoptoitc cells</td></tr><tr><td>&lt;strong&gt;Table of Fly Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Stock center&lt;/strong&gt;</td><td>&lt;strong&gt;Stock ID&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>w&lt;sup&gt;1118&lt;/sup&gt;</td><td></td><td></td><td>Control flies, described in Freeman &lt;em&gt;et al.&lt;/em&gt;, Neuron, 38, 567-580</td></tr><tr><td>drpr&lt;sup&gt;&amp;Delta;5&lt;/sup&gt;</td><td></td><td></td><td>drpr mutant, described in Freeman &lt;em&gt;et al.&lt;/em&gt;, Neuron, 38, 567-580</td></tr><tr><td>Itgbn&lt;sup&gt;2&lt;/sup&gt;</td><td></td><td></td><td>Itgbn mutant, described in Devenport &lt;em&gt;et al.&lt;/em&gt;, Development, 131, 5405-5415</td></tr><tr><td>srpHemoGAL4 UAS-EGFP</td><td></td><td></td><td>described in Br&amp;uuml;ckner &lt;em&gt;et al.&lt;/em&gt;, Dev. Cell., 7, 73-84</td></tr><tr><td>UAS-drpr-IR</td><td>VDRC</td><td>4833</td><td>-</td></tr><tr><td>UAS-Itgbn-IR</td><td>NIG-fly</td><td>1762R-1</td><td>-</td></tr></tbody></table>

phagocytosis assay for apoptotic cells in drosophila embryos

由于其在免疫和炎症难治性疾病中的作用，需要更详细地阐明凋亡细胞吞噬作用的分子机制。我们本文开发了一种使用果蝇果蝇定量研究吞噬作用的实验方法，其中基因网络控制吞噬反应是从哺乳动物进化保守的。为了准确地检测和计数使用全部动物吞噬和吞噬吞噬细胞，将果蝇胚胎匀浆以获得包括吞噬细胞和凋亡细胞的分散细胞。使用分散的胚胎细胞使我们能够测量体内吞噬水平，就像我们进行体外吞噬作用测定一样，可以观察全胚胎中的所有吞噬细胞和凋亡细胞，并精确定量吞噬水平。我们确认这种方法再现了以前的研究确定了凋亡细胞吞噬所需的基因。这种方法允许吞噬死细胞，当与强大的果蝇遗传学结合时，将揭示吞噬细胞凋亡细胞的迁移，识别，吞噬和降解所引起的复杂的吞噬反应。

为了检测凋亡细胞的吞噬作用，收集了发育阶段16的果蝇胚胎，并作为分散细胞制备。血细胞，巨噬细胞果蝇 ，进行免疫细胞化学对血细胞标记"Croquemort" 17，22使用特异性抗体19,21染色，凋亡细胞通过TUNEL在分散的胚胎细胞（ 图1A）染色。 Croctemort，?...

Watch this Scientific Journal Video about Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos at JoVE.com

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

我们在此描述使用果蝇分散的胚胎细胞的吞噬作用。它使我们能够容易且准确地量化体内吞噬水平，并确定凋亡细胞吞噬所需的新分子。

细胞凋亡细胞的吞噬作用<em&gt;果蝇</em&gt;胚胎

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

phagocytosis-assay-for-apoptotic-cells-in-drosophila-embryos

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Cell Biology

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Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

Unit 1

Cell Death

SCIENTISTS IN ACTION

9.8K 次观看.  Kanazawa University. 我们在此描述使用果蝇分散的胚胎细胞的吞噬作用。它使我们能够容易且准确地量化体内吞噬水平，并确定凋亡细胞吞噬所需的新分子。 

视频: Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

神经科学

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

用 CaspaseTracker 生物传感器检测 Anastasis体内

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

医学

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

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  ...

生物学

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

使用 Vivo 法戈西西亚赛评估成人嗜血杆菌黑色粒细胞的年龄特异性噬细胞能力

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

An In Vivo Assay to Quantify Bacterial Phagocytosis in Adult Drosophila Flies

Source: Nazario-Toole, A. E., et al. <a href="https://app.jove.com/v/59543">Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay.</a> J. Vis. Exp. (2019)
This video demonstrates an in vivo assay designed to measure bacterial phagocytosis in adult Drosophila flies. Following the injection of fluorescently labeled bacteria into the abdomen of adult flies, hemocytes associated with the dorsal vessel engage in phagocytosis. The introduction of trypan blue dye serves to quench the fluorescence emitted by extracellular, non-phagocytosed bacteria. This quenching process enhances the identification of phagocytosed bacteria when observed under a fluorescence microscope.

定量成年果蝇细菌吞噬作用的体内测定

1. Prepare Fluorescein particles for injection

	Reconstitute 10 mg of commercially available, heat-killed bacteria particles labeled with fluorescein ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

The Phagocytosis Assay: An Immunostaining Technique to Visualize Apoptotic Cell Engulfment in the Embryonic Cells of Drosophila melanogaster

Source: Nonaka, S. et al. <a href="https://app.jove.com/v/56352">Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos.</a> J. Vis. Exp. (2017).
This video demonstrates an immunostaining technique to visualize apoptotic cell engulfment in the homogenized embryonic cells of Drosophila melanogaster. This method provides insights into cellular physiology by determining the extent of apoptotic cell engulfment.

吞噬作用测定:一种免疫染色技术，用于可视化黑腹果蝇胚胎细胞中的凋亡细胞吞噬

Source: Nonaka, S. et al. Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos. J. Vis. Exp. (2017).
This video demonstrates an immunostaining ...

Biological Techniques

Assay Techniques

Cell-based assays

Fixed Cell Efferocytosis Assay: A Method to Study Efferocytic Uptake of Apoptotic Cells by Macrophages Using Fluorescence Microscopy

Source: Taruc, K., et al. <a href="https://app.jove.com/v/58149">Quantification of Efferocytosis by Single-cell Fluorescence Microscopy.</a> J. Vis. Exp. (2018).
In this video, we demonstrate the efferocytosis assay to study the clearance of apoptotic cells using confocal microscopy. Efferocytosis is a multi-step process where apoptotic cells are engulfed by phagocytes and degraded within vesicles called efferosomes. This protocol provides a method to visualize internalized and non-internalized portions of individual apoptotic cells and quantify efferocytosis.

固定细胞胞吐作用测定:一种使用荧光显微镜研究巨噬细胞对凋亡细胞的泡生细胞摄取的方法

Source: Taruc, K., et al. Quantification of Efferocytosis by Single-cell Fluorescence Microscopy. J. Vis. Exp. (2018).
In this video, we demonstrate the ...

细胞凋亡细胞的吞噬作用

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