We thank Matthew O'Connell, Maryam Jahanshahi, and Andreas Jenny for assistance. We thank Istv&#225;n And&#243; for plasmatocyte-specific antibodies, Utpal Banerjee for dome-meso-EBFP2 flies, Julian Martinez-Agosto for antp&gt;GFP flies, and Michael O'Connor for ptth and ptth&gt;grim flies. These methods were developed with support by the Kimmel Foundation, the Leukemia &amp; Lymphoma Society, NIH/NCI R01CA140451, NSF 1257939, DOD/NFRP W81XWH-14-1-0059, and NIH/NCI T32CA078207.

1.循环血细胞浓度<ol><li> 6小时 - 以获得大致相同的发育阶段的幼虫该测定中，通过允许雌性产卵2的一个固定的时间段限制鸡蛋收集。 </li><li>在解剖盘孔填充用1×磷酸盐缓冲盐水（PBS， 表1）收集幼虫。 </li><li>对于每一个幼虫，将10微升1×PBS中在微量离心管在冰上和10μl1×PBS中在一个干净的解剖垫。将解剖盘上的照明体视显微镜基地。 </li><li>通过将它放置在纸巾上干燥个别幼虫将其转移到解剖垫PBS下降之前擦拭。 </li><li>用钳子，轻轻撕开，小心反转角质层释放淋巴。 注:请注意不要划伤或戳的角质层，因为这可能会释放出无柄血细胞29。 </li><li>用移液管，收集来自解剖垫血淋巴。避免同llecting幼虫的碎片，如肥胖的身体。 </li><li>血淋巴添加到微量离心管中，并通过上下抽吸混合。 注意:几个样品可以一次被收集并在冰上保持长达一个小时。 </li><li>可选:将台盼蓝与等份血淋巴样品染色并排除死细胞。算上加台盼蓝，因为它是细胞毒性的3分钟内的细胞。 </li><li>吹打起来，装载10微升到血球室之前向下混合样品。 </li><li>如果使用自动细胞计数，设置细胞大小和圆适当的参数。例如，设定为2μm的最小单元尺寸为22微米的最大细胞大小，和75的圆- 80％的圆度来检测正常，圆形血细胞34。尝试使用不同的参数来检测更大，纺锤状血细胞，如lamellocytes。或者，使用血球手动计数血细胞。 </li></ol> 2.巡回culating血细胞免疫组化<ol><li> 6小时 - 以获得大致相同的发育阶段的幼虫该测定中，通过允许雌性产卵2的一个固定的时间段限制鸡蛋收集。 </li><li>将一个盖玻片在6-或12孔平板的每个孔中。 注:方形盖玻片（22毫米）配合在6孔板和轮盖玻片（18毫米直径）配合在12孔板中。 </li><li>在解剖盘井充满1X PBS收集幼虫。 </li><li>放置5微升1×PBS中在每个盖玻片的中心。 </li><li>将平板上的照明体视显微镜基地。 </li><li>干上组织一个单独的幼虫擦拭，然后将在PBS盖玻片上。 </li><li>用钳子，轻轻撕开，仔细反转角质层。取出幼虫尸体之前，用它来均匀分布的淋巴。 注:请注意不要划伤或戳的角质层，因为这可能会释放出无柄血细胞29。 </li><li> RepeaT代表所有的幼虫，收集每盖玻片1个幼虫血淋巴。允许血细胞附着在盖玻片5 -在室温下室温8分钟，不要超过固定前30分钟。 </li><li>通过加入5微升7.5％的甲醛（ 表1），以每个盖玻片，在室温下15分钟固定的血细胞。 </li><li>洗涤用1×PBS盖玻片三次。提示板和每次冲洗后吸出PBS。为了防止在下一步骤中的透液的流失，使用抽吸器从盖玻片的外周除去过量的PBS。 </li><li>加入100μl透溶液（ 表1），以每个盖玻片在室温20分钟。 </li><li>提示并轻轻拍打板吸出通透的解决方案。 </li><li>稀释使用根据供应商的规范抗体溶液（ 表1）第一抗体。至少添加500μl的初级抗体溶液至盖玻片在12孔板中。在6孔板中添加至少1.5毫升在4℃孵育过夜。 </li><li>除去第一抗体溶液，并用1×PBS在轨道摇床，三次洗涤盖玻片10分钟。 </li><li>根据供应商的规格稀释二级抗体与抗体溶液。至少添加500μl的第二抗体溶液，以12孔板的盖玻片。添加至少1.5毫升6孔板中。在室温下孵育至少2小时。 </li><li>去除二级抗体溶液并用1×PBS在轨道摇床上洗涤盖玻片10分钟，如在步骤2.10，三次。 </li><li>在洗涤步骤中，干净的玻璃显微镜用组织湿巾70％的乙醇（ 见表1）滑动。对于每个盖玻片，置于载玻片上5微升安装缓冲液（ 表1）。 注:两个盖玻片适合在一张幻灯片。 </li><li>吸出最后的PBS洗涤。 </li><li>使用镊子小心从板和地点，INV取出盖玻片erted，在安装缓冲器的顶部。 注:显微镜载玻片可成像之前被储存在4℃。最大存储时间取决于所用的抗体的稳定性。 6-和12孔板可冲洗和再利用。 </li></ol> 3. 在水晶体内细胞黑化<ol><li>以获得大致相同的发育阶段的幼虫该测定中，通过允许雌性产卵的2-6小时的固定时段限制鸡蛋收集。 </li><li>开始之前，设定在60℃的加热源。 注:60℃，10分钟的热循环仪程序（随后是25℃保持）的效果最好，但水浴或其它加热源是足够只要热量被始终如一地均匀地分布在各幼虫。 </li><li>在解剖盘孔填充用1×PBS（ 表1）收集幼虫。 </li><li>一次一个，干上的组织幼虫在PCR管的底部擦拭和地点。将每个幼虫在一个单独的PCR管。 （ 见图3A。） </li><li>对于一致的结果，保证幼虫留在PCR管的底部在4℃，10心寒的管幼虫 - 15分钟和/或轻轻加热前轻敲管。 </li><li>放置在热循环仪（或水浴）的PCR管。热，在60℃下进行10分钟。 </li><li>小心地从PCR管取出幼虫进入解剖盘井充满了新鲜的1X PBS。 </li><li>上的组织干幼虫擦拭并在立体显微镜下安排在成像平面上。 </li><li>一味地由多个个人得分幼虫的图像。 </li></ol> 4.幼虫淋巴腺免疫组化注:淋巴腺距离上背侧大脑略低于一个幼虫的前端大约三分之一的长度。 （参见图3B中的箭头）。淋巴腺侧翼背船只和最容易解剖附着在口呼KS或到大脑。野生型，三龄淋巴腺是非常小的结构;主波瓣大约100 - 200微米的长度。 （参见图4A）。 <ol><li> 6小时 - 以获得大致相同的发育阶段的幼虫该测定中，通过允许雌性产卵2的一个固定的时间段限制鸡蛋收集。 </li><li>淋巴结清扫术<ol><li>对于每个实验条件下，加入1ml 1×PBS中（ 表1）至24孔板的一个孔中。 </li><li> 0.1％的PBST（ 表1）1滴添加到各孔用一次性移液管。 注:洗涤剂，例如吐温20，被添加以降低表面张力，从而允许组织下沉到井的底部。 </li><li>放置板在冰上平。 </li><li>在解剖盘井充满1X PBS收集幼虫。 </li><li>将干净的解剖盘上的照明体视显微镜基地。使用一次性移液管放置SMaL公司升下降0.01％PBST（ 表1）上的垫。转移一个幼虫解剖一个PBST下降。 </li><li>握住幼虫有一对钳约1/4长度从后端，背侧。 </li><li>使用另一双钳抢到角质层前壁立即向持有幼虫镊子。轻轻一拉角质层朝前端，直到口钩暴露。 注意:目的是要剥离的角质层，而不会干扰任何内部结构。 </li><li>松开角质层，同时使用钳剪幼虫两种。取下PBST降后结束。 注:如果角质层没有一路剥下来了口钩，切幼虫在两个取出后结束。用一对镊子的保持角质层的边缘，并使用另一对钳子的通过开口推口钩。这将反转角质层和揭露口钩。这可以被用来作为一种altern吃了解剖方法为好。 </li><li>用一对镊子的牵制对稳定性的角质层（或者腹面角质层，背侧角质层瓣，或两者）。 </li><li>使用另一双钳抓住露出口钩，轻轻地拉出来。 注意:这将角质层从内部结构分开。理想的情况下，眼睛/触角成虫盘，脑，环腺和淋巴腺侧翼背容器将保持附着在口钩。 </li><li>虽然还拿着口钩，小心地取出多余的结构，如唾液腺，脂肪体和肠。 注:如果大脑从口钩分离，淋巴腺可能仍附在并与大脑收集。在这种情况下，保持腹神经索代替口钩。 </li><li>用口钩或腹神经索作为手柄，转移含有淋巴腺在冰上井的解剖复杂，不要超过30分钟固定前。 </li></ol></li><li>固定和免疫组化<ol><li>放置在立体显微镜基的24孔板。 </li><li>使用P200移液器小心地从井里取出PBST。 注:空，邻井暂时存废有用。 </li><li>轻轻加入200微升3.7％的甲醛（ 表1）向下井的侧面，摇动板以确保解剖组织被完全淹没。 </li><li>返回板冰上30分钟。如果淋巴腺表达荧光蛋白（S），保持覆盖以防止光漂白的板。 </li><li>使用P200移液器小心取出固定液。 </li><li>通过加入200μl的1×PBS中的井和放置板在定轨摇床上5分钟，在室温下洗涤。使用P200移液器小心取出PBS。 注:固定淋巴腺可以在冰留在PBS直到淋巴腺所有实验条件是dissected并固定。 </li><li>重复洗涤步骤两次以上。 </li><li>加入200μl通透溶液（ 表1）到孔中。上在室温45分钟轨道摇床设定的板。 注意:45分钟是"黄金标准"淋巴腺通透但作者都只有20分钟后成功。 </li><li>删除与P200移液器的解决方案。 </li><li>稀释使用根据供应商的规范抗体溶液（ 表1）第一抗体。添加300微升初级抗体溶液至孔，并确保解剖组织被完全淹没。在4℃孵育过夜。 </li><li>使用P200移液器除去初级抗体。 </li><li>通过加入200μl的1×PBS中的井和放置板在定轨摇床上10分钟，在室温下洗涤。使用P200移液器小心取出PBS。 </li><li>重复洗涤步骤两次以上。 </li><li>二次稀释一根据供应商的规格与抗体溶液ntibody。加入300微升二抗溶液至孔，并确保解剖组织被完全淹没。孵育在轨道摇床上于室温至少2小时。 </li><li>使用P200移液器去除二级抗体并在步骤4.3.12＆4.3.13描述洗净。最后一次洗涤后不要取出PBS。 </li></ol></li><li>淋巴腺安装<ol><li>清洁的玻璃显微镜载玻片用70％的乙醇（ 见表1）和组织湿巾。 </li><li>对于每种实验条件下，放置于载玻片上安装缓冲液（ 表1）中的一个的下降。 注:两个条件适合在一张幻灯片。安装缓冲体积取决于待安装淋巴腺的数目。使用2微升约18 - 20淋巴腺，1μl的10 - 12日，和0.5微升5或更小。 </li><li>广场上的照明立体底座的显微镜载玻片。 </li><li>用于一次最多两个条件下，从井传输所有淋巴腺到安装缓冲用钳子，使用口钩或腹神经索作为手柄。 </li><li>空间中的解剖组织均匀的圆形或矩形形状，扩展的过程中安装缓冲器。 </li><li>对于每个解剖组织，独立，背船只下滑动钳一个夹钳和朝向安装缓冲外围轻轻一拉。 注意:这将引起淋巴从解剖组织的其余部分进行压盖和扁平玻璃上的淋巴结。 </li><li>使用镊子的一个夹钳和锯切运动，切淋巴腺和大脑之间的背血管。 </li><li>移动至解剖组织的其余部分淋巴腺的相对侧，在缓冲的最外边缘。 注:不需要解剖组织最终将形成围绕淋巴腺周边并作为对whic支持小时盖玻片将休息。 </li><li>重复，直到所有的淋巴腺从解剖组织分离，保留不需要的剖分组织在中心放置的一个。 </li><li>以两个手指之间的盖玻片。检查盖玻片是免费的灰尘和指纹。如有必要，使用纸巾擦拭和70％的乙醇清洁盖玻片。 </li><li>确保盖玻片的边缘平行于玻璃载片的边缘，小心地放在安装缓冲器盖玻片。 注:显微镜载玻片可成像之前被储存在4℃。最大存储时间取决于所用的抗体的稳定性。 24孔板可冲洗和再利用。 </li></ol></li></ol> 5.成像<ol><li>固定图像和血细胞根据制造商的操作手册在20X或更高的放大倍率装在一个适当的标准荧光或共聚焦显微镜淋巴腺。在支架上的图像整体的幼虫根据制造商的操作手册以2倍的放大倍率ARD体视显微镜。 </li><li>按照软件供应商的说明去卷积，如果需要的话。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>解</td><td>组成</td><td>存储</td><td>注释</td></tr><tr><td rowspan="5"> 1X PBS </td><td> 200毫克/升的氯化钾</td><td rowspan="5">室内温度</td><td rowspan="5"></td></tr><tr><td> 200毫克/升磷酸二氢钾</td></tr><tr><td> 8000毫克/升氯化钠</td></tr><tr><td> 1150毫克/升的磷酸氢二钠</td></tr><tr><td>卫生署2 O </td></tr><tr><td>固色剂</td><td> 3.7％或7.5％的甲醛在1X PBS </td><td>室温在黑暗中</td><td>甲醛是有毒的。 </td></tr><tr><td rowspan="3">通透的解决方案/抗体稀释剂</td><td> 0.4％的Triton </td><td rowspan="3"> 4℃ </td><td rowspan="3">标准公式使用0.4％的Triton但作者使用0.1％吐温20以成功。使用根据供应商的推荐浓度稀释小学和中学的抗体。 </td></tr><tr><td> 5％牛血清白蛋白，正常山羊血清，或正常驴血清</td></tr><tr><td> 1X PBS </td></tr><tr><td> 70％的乙醇</td><td>蒸馏水中的70％的200度乙醇</td><td>室内温度</td><td></td></tr><tr><td rowspan="4">安装缓冲</td><td> 0.5％N-丙基没食子酸</td><td rowspan="4"> 4℃在黑暗中</td><td rowspan="4">棓酸正丙酯是有害的。 DAPI是一种诱变剂。 </td></tr><tr><td> 80％甘油</td></tr><tr><td>可选:1μg/ ml的DAPI（4&#39;，6-二脒基-2-苯基） </td></tr><tr><td> 1X PBS </td></tr><tr><td> 0.1％PBST </td><td> 0.1％吐温20在1×PBS中</td><td>室内温度</TD&gt; <td></td></tr><tr><td> 0.01％PBST </td><td> 1:10稀释0.1％的PBST </td><td>室内温度</td><td></td></tr></tbody></table> 表1.本议定书中所使用的解决方案。

<ol>
	<li>Evans, C. J., Hartenstein, V., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thicker+than+blood:+conserved+mechanisms+in+Drosophila+and+vertebrate+hematopoiesis.">Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis.</a> Dev Cell. 5 (5), 673-690 (2003).</li><li>Crozatier, M., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+haematopoiesis.">Drosophila haematopoiesis.</a> Cell Microbiol. 9 (5), 1117-1126 (2007).</li><li>Crozatier, M., Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila:+a+model+for+studying+genetic+and+molecular+aspects+of+haematopoiesis+and+associated+leukaemias.">Drosophila: a model for studying genetic and molecular aspects of haematopoiesis and associated leukaemias.</a> Dis Model Mech. 4 (4), 439-445 (2011).</li><li>Gold, K. S., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+the+two+myeloid+blood+cell+systems+in+vertebrates.">Drosophila as a model for the two myeloid blood cell systems in vertebrates.</a> Exp Hematol. 42 (8), 717-727 (2014).</li><li>Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+cells+and+blood+cell+development+in+the+animal+kingdom.">Blood cells and blood cell development in the animal kingdom.</a> Annu Rev Cell Dev Biol. 22, 677-712 (2006).</li><li>Ghosh, S., Singh, A., Mandal, S., Mandal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+hematopoietic+hubs+in+Drosophila+adults+generate+hemocytes+and+contribute+to+immune+response.">Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response.</a> Dev Cell. 33 (4), 478-488 (2015).</li><li>Leitao, A. B., Sucena, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+sessile+hemocyte+clusters+are+true+hematopoietic+tissues+that+regulate+larval+blood+cell+differentiation.">Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation.</a> Elife. 4, (2015).</li><li>Makhijani, K., Alexander, B., Tanaka, T., Rulifson, E., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+peripheral+nervous+system+supports+blood+cell+homing+and+survival+in+the+Drosophila+larva.">The peripheral nervous system supports blood cell homing and survival in the Drosophila larva.</a> Development. 138 (24), 5379-5391 (2011).</li><li>Crozatier, M., Ubeda, J. M., Vincent, A., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+immune+response+to+parasitization+in+Drosophila+requires+the+EBF+orthologue+collier.">Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier.</a> PLoS Biol. 2 (8), 196 (2004).</li><li>Markus, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sessile+hemocytes+as+a+hematopoietic+compartment+in+Drosophila+melanogaster.">Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster.</a> Proc Natl Acad Sci U S A. 106 (12), 4805-4809 (2009).</li><li>Minakhina, S., Steward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanotic+mutants+in+Drosophila:+pathways+and+phenotypes.">Melanotic mutants in Drosophila: pathways and phenotypes.</a> Genetics. 174 (1), 253-263 (2006).</li><li>Bina, S., Wright, V. M., Fisher, K. H., Milo, M., Zeidler, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+targets+of+Drosophila+JAK/STAT+pathway+signalling+as+effectors+of+haematopoietic+tumour+formation.">Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation.</a> EMBO Rep. 11 (3), 201-207 (2010).</li><li>Avet-Rochex, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+RNA+interference+screen+identifies+gene+networks+controlling+Drosophila+melanogaster+blood+cell+homeostasis.">An in vivo RNA interference screen identifies gene networks controlling Drosophila melanogaster blood cell homeostasis.</a> BMC Dev Biol. 10, 65 (2010).</li><li>Rodriguez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+immune+system+and+response+genes,+and+novel+mutations+causing+melanotic+tumor+formation+in+Drosophila+melanogaster.">Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster.</a> Genetics. 143 (2), 929-940 (1996).</li><li>Mandal, L., Banerjee, U., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+fruit+fly+hemangioblast+and+similarities+between+lymph-gland+hematopoiesis+in+fruit+fly+and+mammal+aorta-gonadal-mesonephros+mesoderm.">Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm.</a> Nat Genet. 36 (9), 1019-1023 (2004).</li><li>Grigorian, M., Mandal, L., Hakimi, M., Ortiz, I., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+convergence+of+Notch+and+MAPK+signaling+specifies+the+blood+progenitor+fate+in+the+Drosophila+mesoderm.">The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm.</a> Dev Biol. 353 (1), 105-118 (2011).</li><li>Lebestky, T., Jung, S. H., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Serrate-expressing+signaling+center+controls+Drosophila+hematopoiesis.">A Serrate-expressing signaling center controls Drosophila hematopoiesis.</a> Genes Dev. 17 (3), 348-353 (2003).</li><li>Krzemien, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+blood+cell+homeostasis+in+Drosophila+larvae+by+the+posterior+signalling+centre.">Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre.</a> Nature. 446 (7133), 325-328 (2007).</li><li>Mandal, L., Martinez-Agosto, J. A., Evans, C. J., Hartenstein, V., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hedgehog-+and+Antennapedia-dependent+niche+maintains+Drosophila+haematopoietic+precursors.">A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors.</a> Nature. 446 (7133), 320-324 (2007).</li><li>Benmimoun, B., Polesello, C., Haenlin, M., Waltzer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EBF+transcription+factor+Collier+directly+promotes+Drosophila+blood+cell+progenitor+maintenance+independently+of+the+niche.">The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche.</a> Proc Natl Acad Sci U S A. 112 (29), 9052-9057 (2015).</li><li>Sinenko, S. A., Mandal, L., Martinez-Agosto, J. A., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+role+of+wingless+signaling+in+stem-like+hematopoietic+precursor+maintenance+in+Drosophila.">Dual role of wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila.</a> Dev Cell. 16 (5), 756-763 (2009).</li><li>Pennetier, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+control+of+the+Drosophila+hematopoietic+niche+by+bone+morphogenetic+protein+signaling+reveals+parallels+with+mammals.">Size control of the Drosophila hematopoietic niche by bone morphogenetic protein signaling reveals parallels with mammals.</a> Proc Natl Acad Sci U S A. 109 (9), 3389-3394 (2012).</li><li>Dragojlovic-Munther, M., Martinez-Agosto, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix-modulated+Heartless+signaling+in+Drosophila+blood+progenitors+regulates+their+differentiation+via+a+Ras/ETS/FOG+pathway+and+target+of+rapamycin+function.">Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function.</a> Dev Biol. 384 (2), 313-330 (2013).</li><li>Ferguson, G. B., Martinez-Agosto, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yorkie+and+Scalloped+signaling+regulates+Notch-dependent+lineage+specification+during+Drosophila+hematopoiesis.">Yorkie and Scalloped signaling regulates Notch-dependent lineage specification during Drosophila hematopoiesis.</a> Curr Biol. 24 (22), 2665-2672 (2014).</li><li>Milton, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hippo+pathway+regulates+hematopoiesis+in+Drosophila+melanogaster.">The Hippo pathway regulates hematopoiesis in Drosophila melanogaster.</a> Curr Biol. 24 (22), 2673-2680 (2014).</li><li>Evans, C. J., Liu, T., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+hematopoiesis:+Markers+and+methods+for+molecular+genetic+analysis.">Drosophila hematopoiesis: Markers and methods for molecular genetic analysis.</a> Methods. 68 (1), 242-251 (2014).</li><li>Neyen, C., Bretscher, A. J., Binggeli, O., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+study+Drosophila+immunity.">Methods to study Drosophila immunity.</a> Methods. 68 (1), 116-128 (2014).</li><li>Small, C., Paddibhatla, I., Rajwani, R., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+parasitic+wasps+of+Drosophila+and+the+antiparasite+immune+response.">An introduction to parasitic wasps of Drosophila and the antiparasite immune response.</a> J Vis Exp. (63), e3347 (2012).</li><li>Petraki, S., Alexander, B., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+Blood+Cell+Populations+of+the+Drosophila+melanogaster+Larva.">Assaying Blood Cell Populations of the Drosophila melanogaster Larva.</a> J Vis Exp. (105), (2015).</li><li>Rizki, M. T. M., Rizki, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+the+crystal+cells+in+the+larva+of+Drosophila+mekmogaster.">Functional significance of the crystal cells in the larva of Drosophila mekmogaster.</a> Journal of Biophysical and Biochemical Cytology. 5, 235-240 (1959).</li><li>Kurucz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+Drosophila+hemocyte+subsets+by+cell-type+specific+antigens.">Definition of Drosophila hemocyte subsets by cell-type specific antigens.</a> Acta Biol Hung. 58, 95-111 (2007).</li><li>Jung, S. H., Evans, C. J., Uemura, C., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+lymph+gland+as+a+developmental+model+of+hematopoiesis.">The Drosophila lymph gland as a developmental model of hematopoiesis.</a> Development. 132 (11), 2521-2533 (2005).</li><li>Krzemien, J., Crozatier, M., Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+Drosophila+larval+hematopoietic+organ,+hemocyte+homeostasis+and+the+dedicated+cellular+immune+response+to+parasitism.">Ontogeny of the Drosophila larval hematopoietic organ, hemocyte homeostasis and the dedicated cellular immune response to parasitism.</a> Int J Dev Biol. 54 (6-7), 1117-1125 (2010).</li><li>Rizki, T. M., Rizki, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+Larval+Hemocytes+of+Drosophila-Melanogaster.">Properties of the Larval Hemocytes of Drosophila-Melanogaster.</a> Experientia. 36 (10), 1223-1226 (1980).</li><li>Lanot, R., Zachary, D., Holder, F., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembryonic+hematopoiesis+in+Drosophila.">Postembryonic hematopoiesis in Drosophila.</a> Dev Biol. 230 (2), 243-257 (2001).</li><li>McBrayer, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prothoracicotropic+hormone+regulates+developmental+timing+and+body+size+in+Drosophila.">Prothoracicotropic hormone regulates developmental timing and body size in Drosophila.</a> Dev Cell. 13 (6), 857-871 (2007).</li><li>Reimels, T. A., Pfleger, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Rabex-5+restricts+Notch+activity+in+hematopoietic+cells+and+maintains+hematopoietic+homeostasis.">Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis.</a> J Cell Sci. 128 (24), 4512-4525 (2015).</li></ol>

作者宣称，他们没有竞争或经济利益。

在遗传或环境的改变，这里所描述的四种方法可单独或联合用于分析诸如信令，存活，增殖和分化的造血过程中不同的过程果蝇造血是一个动态过程。每只动物血细胞的数量增加35和发育过程中的淋巴腺的结构和基因表达的变化32。之前执行这些测定法，因此，它是通过使雌性产卵的固定时间量，并确认所期望的幼虫的发育阶段来限制蛋集合的关键。对于更短的蛋集合（小?...

调节转录因子和信号转导通路即坐标造血系统的在血液系统疾病的发展，而且故障复杂机制仍然知之甚少。这些转录因子和信号通路，以及它们的调节，是高度果蝇和哺乳动物造血1-5之间是保守的。因此， 果蝇造血系统代表一个优秀的遗传模型来定义的分子机制控制造血和潜在的血液系统疾病。 哺乳动物类似， 果蝇产生在空间和时间上不同的造血的相位血细胞，称为血细胞。传统上， 果蝇造血被认为在胚胎中胚层和在幼虫淋巴腺被限制在相。最近的研究提供的证据表明，血细胞生成，也会发生在幼虫无梗CLU讲演者，并在成人腹部6-8。所有造血阶段产生两种类型的血细胞成熟的:浆细胞和晶体细胞。浆细胞是参与吞噬作用，先天免疫，和伤口愈合的巨噬细胞样细胞。水晶细胞含有黑色素，在昆虫免疫反应和伤口愈合使用的反应所需的亲phenoloxidases。幼虫造血可以产生第三成熟血细胞类型，称为lamellocyte，响应于如寄生蜂感染9,10-某些免疫挑战。 Lamellocytes是功能，与浆细胞和水晶结合的细胞，封装和消除在果蝇幼虫黄蜂奠定鸡蛋大，贴壁细胞。在不存在寄生的，lamellocytes未在野生型幼虫找到。黑色素群众像白化，封装蜂卵;许多果蝇突变株培养在没有寄生的黑色素群众。 lamello的存在核细胞和/或黑色素群众可以指示造血异常。事实上，黑色素质量表型已经被用于鉴定参与造血11-14基因和途径。 幼体造血系统是最广泛研究日期。它是由血细胞在血淋巴中循环的，角质层下图案化无梗血细胞群集，和血细胞驻留在淋巴腺。淋巴腺是一系列附连到背脉管双边瓣。淋巴腺的每个主叶被分成三个主要区域。最外面的区被称为皮层区和包含成熟的血细胞。最里面的区域被称为髓区并且包括静态血细胞前体。第三区，后信令中心，是在淋巴腺充当一个干细胞样的龛的基部一小群细胞。早期的工作建立关键功能的缺口15-18 </suP&gt;，刺猬19,20，JAK-STAT 18，和无翅21的活动来调节幼虫淋巴腺发展。最近的研究已经表明，BMP 22，FGF-RAS 23和河马24,25信令也幼虫淋巴腺内起作用。 这里概述四幼虫造血测定法描述1）测量循环血细胞浓度，定义为每单位体积的细胞数，2）分离和定影循环血细胞对于免疫组化，3）可视晶体细胞在体内 ，和4）解剖，定影，和安装淋巴腺进行免疫组化。这些测定可用于造血读数来评估的功能和在幼虫造血系统的信号通路的规定。虽然这些方法已经在该领域以前使用的，这些试验的视觉文件已经开始最近才8,26-30。这里列举了几个出版物帮助FUL资源描述类似的方法和造血标记26,31-33。此外，控制和海盗是淋巴腺基底膜的有效指标。

<table><tbody><tr><td>PBS tablets</td><td>MP Biomedicals</td><td>2810305</td><td></td></tr><tr><td>dissecting dish</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>microcentrifuge tube</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>silicone dissecting pad, made from Sylgard 184 kit</td><td>Krayden (distributed through Fisher)</td><td>NC9644388 (Fisher catalog number)</td><td>Made in petri dish by mixing components of Sylgard elastomer kit according to manufacturer instructions.</td></tr><tr><td>stereomicroscope</td><td>Morrell Instruments (Nikon distributor)</td><td>mna42000, mma36300</td><td>Nikon models SMZ1000 and SMZ645</td></tr><tr><td>tissue wipe</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>forceps</td><td>Electron Microscopy Sciences</td><td>72700-DZ</td><td></td></tr><tr><td>p200 pipette</td><td>Eppendorf</td><td>3120000054</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Countess cell counting chamber slides</td><td>Invitrogen</td><td>C10283</td><td></td></tr><tr><td>hemocytometer</td><td>Hausser Scientific</td><td>3200</td><td></td></tr><tr><td>trypan blue stain</td><td>Life Technologies</td><td>T10282</td><td></td></tr><tr><td>formaldehyde</td><td>Fisher</td><td>BP531-500</td><td></td></tr><tr><td>Triton</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Tween 20</td><td>Fisher</td><td>BP337-500</td><td></td></tr><tr><td>bovine serum albumin</td><td>Rocky Mountain Biologicals</td><td>BSA-BSH-01K</td><td></td></tr><tr><td>normal goat serum</td><td>Sigma</td><td>G9023-10ML</td><td></td></tr><tr><td>normal donkey serum</td><td>Sigma</td><td>D9663-10ML</td><td></td></tr><tr><td>200 proof ethanol</td><td>VWR</td><td>V1001</td><td></td></tr><tr><td>N-propyl gallate</td><td>MP Biomedicals</td><td>102747</td><td></td></tr><tr><td>glycerol</td><td>VWR</td><td>EM-4750</td><td></td></tr><tr><td>DAPI (4&amp;rsquo;,6-diamidino-2-phenylindole)</td><td>Fisher</td><td>62248</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>351146</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>351143</td><td></td></tr><tr><td>microscope cover glass, 22 mm square</td><td>Fisher</td><td>12-544-10</td><td></td></tr><tr><td>microscope cover glass, 18 mm circular</td><td>Fisher</td><td>12-545-100</td><td></td></tr><tr><td>glass microscope slides</td><td>Fisher</td><td>22-034-980</td><td></td></tr><tr><td>thermal cycler</td><td>Eppendorf</td><td>E950010037</td><td>Mastercycler EP Gradient S</td></tr><tr><td>PCR tubes</td><td>USA Scientific</td><td>1402-2700</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>351147</td><td></td></tr><tr><td>disposable transfer pipet</td><td>Fisher</td><td>13-711-9AM</td><td></td></tr><tr><td>fluorescence microscope</td><td>Zeiss</td><td></td><td>Axio Imager.Z1</td></tr></tbody></table>

methods to examine the lymph gland and hemocytes in drosophila larvae

果蝇 果蝇和哺乳动物造血系统之间有许多共同点存在，即使果蝇缺乏淋巴系表征哺乳动物适应性免疫，和哺乳动物造血发生在时间和空间上不同的阶段产生多种血细胞谱系。这两个系统都保持血液祖细胞与扩大或更换成熟谱系的水库。造血系统允许果蝇和哺乳动物以应对和适应免疫的挑战。重要的是，用于控制生成，维护，和造血系统的功能的转录调节和信号传导途径是从果蝇保守到哺乳动物。这些相似性允许果蝇被用于遗传模型造血发育和疾病。 下面我们详细分析研究果蝇幼虫的造血系统。在特别是，我们概括的方法来测量血细胞数量和浓度，体内可视化的特定成熟细胞系，并在循环和造血器官进行对血细胞免疫组化。这些分析可以揭示基因表达的变化的细胞过程，包括信令，存活，增殖和分化，并且可以用于研究各种关于造血的问题。在果蝇中可用的遗传工具相结合，这些测定法可以用于评估在所定义的遗传改变的造血系统。尽管没有特别这里概述，这些试验也可以用来检查环境的改变，如感染或饮食的影响，对造血系统。

循环血细胞浓度血细胞数在整个幼虫发育35增加。为了说明，该方法检测血细胞数量和浓度的差异，无论生物原因，我们测量延迟和非延迟的幼虫血细胞的浓度。促前胸腺激素（PTTH）由PTTH产生神经元（PTTH&gt;严峻）的遗传消融的损失产生于幼虫发育36的延迟。对于每个基因型，对于在25℃升高至少8个人幼虫在方案...

Watch this Scientific Journal Video about Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae at JoVE.com

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

果蝇和哺乳动物造血系统有许多共同的特点，使果蝇有吸引力的遗传模型来研究造血功能。这里，我们证明了免疫组化的主要造血幼虫器官解剖和安装。我们还描述的方法来测定各种幼虫造血车厢，包括循环血细胞和无柄水晶细胞。

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

methods-to-examine-the-lymph-gland-and-hemocytes-in-drosophila-larvae

Research

JoVE Journal

Developmental Biology

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

12.7K 次观看.  The Icahn School of Medicine at Mount Sinai.  果蝇和哺乳动物造血系统有许多共同的特点，使果蝇有吸引力的遗传模型来研究造血功能。这里，我们证明了免疫组化的主要造血幼虫器官解剖和安装。我们还描述的方法来测定各种幼虫造血车厢，包括循环血细胞和无柄水晶细胞。 

视频: Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

科研

发育生物学

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

免疫与感染

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

神经科学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

生物学

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

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 ...

Drosophila Larval IHC

Immunohistochemistry (IHC) is a technique used to visualize the presence and location of proteins within tissues. Drosophila larvae are particularly amenable to IHC because of the ease with which they can be processed for staining. Additionally, the larvae are transparent, meaning that some tissues can be visualized without the need for dissection.
In IHC, proteins are ultimately detected with antibodies that specifically bind to &ldquo;epitopes&rdquo; within the protein of interest. In order to preserve these epitopes, tissues must be fixed prior to staining. Furthermore, in order for antibodies to penetrate membranes, cells must be permeabilized with detergents. This video article offers a detailed view of the reagents, tools, and procedures necessary for the staining of dissected larval tissues, including fixation, blocking, and staining steps. Also featured is a demonstration of tissue mounting techniques for fluorescence microscopy. Finally, examples of the broad range of applications for these techniques (and some variations upon them) are provided.

Drosophila melanogaster Larval Immunohistochemistry IHC

果蝇幼虫的免疫组化

Immunohistochemistry (IHC) is a technique used to visualize the presence and location of proteins within tissues. Drosophila larvae are particularly ...

教育

JoVE Science Education

基础生物学

模式生物I:酵母，果蝇和秀丽线虫

Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www.jove.com/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
The Drosophila hemolymph serves as both blood and interstitial fluid, circulating throughout the body during the larva and adult stages. This video describes a protocol to collect larval hemolymph in order to quantify the circulating cells.

角质层破坏:一种从果蝇幼虫中收集血淋巴的方法

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
The Drosophila ...

JoVE Encyclopedia of Experiments

黑腹果蝇（果蝇）

幼虫

Lipid Droplet Staining of Larval Drosophila Oenocytes: A Technique for Assessment of Lipid Droplet Accumulation Under Normal and Stressed Condition Using BODIPY-Based Dye

Source: Wei, C., et al., <a href="https://www.jove.com/v/60606">Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae.</a> J. Vis. Exp. (2019).
This video demonstrates lipid droplet staining in Drosophila larval oenocytes - hepatocyte-like cells - using the lipophilic fluorescent BODIPY based dye. These cells act as a model for studying lipid metabolism during development and in response to environmental stress such as starvation.

果蝇幼虫油滴红细胞的脂滴染色:一种使用 BODIPY 基染料评估正常和应激条件下脂滴积累的技术

Source: Wei, C., et al., Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae. J. Vis. Exp. (2019).
This video demonstrates lipid ...

Biological Techniques

Staining Techniques

Cell staining

Larval Lymph Gland Dissection: Isolating the Drosophila Hemocyte-Producing Organ

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www.jove.com/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
This video describes the Drosophila lymph gland&#8212;the primary site of hematopoiesis in the fly larva&#8212;and shows an example protocol to dissect and mount the organ for immunohistochemistry and fluorescent imaging.

幼虫淋巴腺清扫术:分离果蝇血细胞产生器官

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
This video describes ...

方法来检查淋巴腺和血细胞中

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在幼虫馈电电路的功能分析果蝇