我们感谢罗伯特 Heinzen 博士提供了 mCherry 表达贝柯克斯柯克斯的股票。我们感谢路易斯特谢拉博士提供的无脊椎动物彩虹病毒6和布卢明顿股票中心提供飞行股票。该项目部分由 NIH 赠款 R00 AI106963 (A.G.G.) 和华盛顿州立大学资助。

1.体内感染
<ol><li>介质和设备<ol>
<li>在无菌条件下, 准备新鲜的果蝇全血细胞隔离培养基 (DHIM), 含有75% 施耐德的果蝇培养基, 25% 胎牛血清 (血清) 和过滤消毒。</li>
		<li>层2-3 片10厘米 x 10 厘米的石蜡膜下显微镜。</li>
		<li>准备玻璃毛细管。将毛细管拉拔加热器设置为最大值的55%。将毛细管拉到大约10µm 的尖点。</li>
		<li>用矿物油回填毛细管。</li>
		<li>将填充的毛细管装入 nanoinjector (图 1A), 并通过使用镊子断开尖端 (图 1B) 打开融合的毛细管尖端。尖端的外径应为100µm, 便于包囊的摄取。</li>
		<li>从毛细管尖端排出尽可能多的油, 然后用 DHIM 填充。为方便地可视化的血淋巴和油的边界, 包括在油和 DHIM 之间的气泡 (图 1B)。</li>
	</ol>
</li>
	<li>血淋巴提取<ol>
<li>从食品瓶内壁中选取 3rd龄果蝇幼虫, 轻轻使用镊子, 将其放入100µm 过滤器 (图 1C)。3rd龄幼虫在成年雌性产卵后3-6 天内被发现。 注: 这些实验使用了基因型, w1118;p {w+ mC= Hml-GAL4. Δ} 2, P {w+ mC= 无人参与-2 xegfp} AH2, 因为包囊从这些动物表达增强的绿色荧光蛋白 (EGFP) 帮助在细胞的鉴定通过显微镜。</li>
		<li>在幼虫上倒入5毫升的不育水, 并摇动过滤器5秒. 将滤网放到任务擦除上, 以删除多余的水 (图 1C)。</li>
		<li>将幼虫转化为1.5 毫升离心管。将它们与 CO2气体一起麻醉5秒 (图 1D)。</li>
		<li>将幼虫放在显微镜下的石蜡膜上, 背侧朝上 (图 2A)。</li>
		<li>将玻璃毛细管轻轻地放到幼虫后体上, 以保持它到位, 并扰乱后角质层开放使用精细尖钳 (图 2B)。</li>
		<li>允许血淋巴流到石蜡膜上 (图 2C)。</li>
		<li>在石蜡膜上一次制作一个血淋巴池, 包括包囊20-50 幼虫。</li>
		<li>使用 nanoinjector 上的玻璃毛细管 (图 2D) 进行池内血淋巴。 注: 血淋巴大约有10-20 µL。</li>
		<li>将血淋巴喷射到含有500µL DHIM 的1.5 毫升离心管 (图 2E)。</li>
		<li>重复步骤 1.2.4)-1.2.9) 为每一批幼虫。</li>
	</ol>
</li>
	<li>计算包囊的数目。<ol>
<li>吸管5µL 0.4% 台盼蓝溶液在0.6 毫升离心管染色的死细胞。用吸管轻轻地将 DHIM 和包囊在1.5 毫升管中混合, 并将 DHIM 包括包囊的5µL 转移到0.6 毫升离心管中, 并轻轻混合。</li>
		<li>吸管10µL 细胞从1:1 台盼蓝: 全血细胞混合物进入 hemocytometer。</li>
		<li>计算在 hemocytomter 的4个角场中, 不沾有台盼蓝的活包囊数, 并用公式计算每毫升包囊的浓度: <img alt="Equation 1" src="/files/ftp_upload/57077/57077eq1.jpg"> 其中 X 是每毫升活包囊的浓度;a、b、c 和 d 是 hemocytometer 中计算的每4个字段中的活细胞数 (由台盼蓝排除所确定)。分裂的细胞总数的2是由于1:1 稀释的细胞与台盼蓝。用台盼蓝染色的细胞被认为是死的。</li>
	</ol>
</li>
	<li>
前体感染<ol>
<li>根据每个实验所需的 timepoints 和生物复制量确定要与细胞播种的井数。5.0×104包囊是理想的 RNA 和蛋白质纯化后感染。</li>
		<li>使用以下公式计算感染 DHIM 所稀释的病原体存量: <img alt="Equation 2" src="/files/ftp_upload/57077/57077eq2.jpg"> 感染多样性 (语言) 是每个细胞所需的病毒或细菌的数量。 注: 使用的语言取决于个别的实验和化验结果。这里, 使用了10个基因组等价 (GE)/ C. 柯克斯的细胞, 10 CFU/细胞的李斯特菌, 或 1 TCID50/IIV6 细胞。</li>
		<li>通过将适当的 DHIM 体积添加到管中的病毒或细菌体积中, 为24井板的每一个井准备500µL 病原体培养基。</li>
		<li>在24井板的井中放置一个12毫米圆盖玻璃 (不1厚度)。</li>
		<li>将 DHIM 包括包囊到24井板的井中。</li>
		<li>在井中添加500µL 病原体培养基包囊。</li>
		<li>离心板在 1000 x g 5 分钟。</li>
		<li>在28摄氏度孵育1小时的盘子。每15分钟, 轻轻地将盘子从后面倾斜, 然后从左向右5秒。</li>
		<li>在 1 h 的入侵/附着步骤 1.4.8), 轻轻地吸走病菌培养基和洗涤包囊与新鲜的 DHIM, 并补充它与500µL 新鲜 DHIM。</li>
		<li>在所需时间内孵化受感染的包囊。在这些实验中,柯克斯-或 IIV6-infected 包囊被孵化24小时, 而李斯特菌感染的包囊则孵化为1、2或4小时。</li>
	</ol>
</li>
</ol>2.体内感染
<ol><li>感染<ol>
<li>在室温下加热果蝇果汁琼脂板15分钟, 如前所述20。<ol>
<li>添加30克琼脂到700毫升的水和高压釜40分钟。</li>
			<li>将0.5 克甲基 paraben 在10毫升的绝对乙醇中溶解。</li>
			<li>将甲基 paraben 溶液加入300毫升果汁浓缩液中。</li>
			<li>快速将果汁浓缩成蒸压琼脂溶液, 并将5毫升放入 10 x 35 毫米培养皿中。</li>
			<li>在板材冷却了15分钟之后, 将它们存储在4摄氏度。</li>
		</ol>
</li>
		<li>在步骤1.2.1 后准备 3rd龄幼虫-1.2.3)。</li>
		<li>将酵母糊放在琼脂盘上。在琼脂盘中进行细切口, 幼虫可以迁移以避免干燥 (图 3a)。用石蜡膜 (图 3B、C) 组装0.001 毫米尖钨针与持有钳。</li>
		<li>吸管50µL 高效价 mCherry 表达-柯克斯(5.95×109 GE/mL) 到石蜡膜下的立体显微镜下, 并将幼虫放入细菌池中。</li>
		<li>将幼虫放入细菌池中。用钨针刺出幼虫 (图 3D)。将幼虫转移到琼脂盘上 (图 3E)。</li>
		<li>将剩余的病原体转移到琼脂板上, 用石蜡膜封住钢板。</li>
		<li>将幼虫放在潮湿的空气中, 直到感染后所需的时间 (图 3F)。在这个实验中,柯克斯感染的幼虫在盘子上24小时。</li>
	</ol>
</li>
	<li>包囊的血淋巴提取与电镀<ol>
<li>在步骤1.1 之后准备介质和设备。</li>
		<li>在24井板的井中放置一个12毫米圆盖玻璃 (不1厚度)。吸管500µL DHIM 入井。</li>
		<li>从受感染的幼虫中提取血淋巴 1.2.4)-1.2.8)。</li>
		<li>将血淋巴喷射至井下步骤 2.2.2)。</li>
		<li>重复 2.2.3) 和 2.2.4) 为多批次的幼虫。</li>
		<li>离心板在 1000 x g 5 分钟。</li>
	</ol>
</li>
</ol>3. 可视化
<ol><li>固定和染色<ol>
<li>在允许包囊在井中的圆盖玻璃上定居后, 轻轻地从每个井中取出介质。</li>
		<li>轻轻地添加200µL 4% 多聚甲醛 (粉煤灰) 的每个井落户包囊。在室温下孵化包囊20分钟。</li>
		<li>去除4% 的粉煤灰, 轻轻地添加200µL 的 PBS, 含有0.1% 的海卫 X-100 和1% 牛血清白蛋白 (BSA) 的每一个井。在室温下孵化包囊10分钟。</li>
		<li>卸下 PBS 并轻轻地将 1× 4 '、6-diamidino-2 苯基吲哚 (DAPI) 的200µL 添加到每个井中。室温下在黑暗中孵化包囊10分钟。</li>
		<li>移除 DAPI 解决方案, 并轻轻地将 PBS 添加到每个井中。在室温下孵化包囊5分钟。</li>
		<li>在玻璃显微镜滑梯上滴下10µL 的 antifade 安装介质。</li>
		<li>从每个井中取出 PBS 后, 用细尖钳将盖玻璃从24井板上取下。轻轻地将盖子玻璃放在玻璃滑梯上的 antifade 安装介质上, 包囊朝下。</li>
		<li>让幻灯片干燥, 把它放在黑暗中过夜。</li>
	</ol>
</li>
	<li>共焦成像<ol>
<li>为 DAPI、EGFP 和 mCherry 的三色成像配置共焦显微镜。使用以下设置: DAPI 励磁 (前) 405 毫微米, 发射 (em) 415-480 毫微米;EGFP 前488毫微米, em 493-564 毫微米;mCherry 前587毫微米, em 597-700 毫微米。</li>
		<li>将样品放在显微镜上, 用 63 x/1.4 数值孔径 (NA) 的目标聚焦在样品上。在图像的视野中找到所需的包囊。</li>
		<li>调整激光功率和探测器增益, 以达到适当的样品曝光。检查多个 z 平面以确保曝光级别适合整个采样厚度。</li>
		<li>在整个全血细胞的 z 轴上找到顶部和底部位置。将这些位置设置为 z 切片的起始和结束位置。</li>
		<li>仅使用扫描缩放来图像包含全血细胞的区域。通常使用3X 的缩放系数。</li>
		<li>在相应分辨率下收集图像系列, 如 x y 平面中的 1024 x 1024 像素, 以及 z 维度中的0.3 µm 间距。</li>
	</ol>
</li>
	<li>3D 模型重建 注意: 开放源码软件存在, 执行下面描述的3D 模型重建的许多功能。<ol>
<li>将 z 切片图像系列文件导入与共焦显微镜相关的软件, 用于3D 模型重建。</li>
		<li>选择一个单元格, 显示与 DAPI 和 mCherry 一起染色的细胞核, 表示全血细胞的 EGFP 中表达柯克斯。裁剪图像系列以仅包含单个单元格。</li>
		<li>选择使用软件的预打包算法重新构造3D 模型的3D 查看器选项。在混合、曲面和混合选项之间选择所需的3D 表示形式。在此方法中, 使用曲面模型显示包囊、原子核和柯克斯。</li>
		<li>通过按住鼠标按钮并在屏幕周围拖动光标, 观察来自不同观察位置的3D 重建细胞。调整模型光源的单元方向和位置, 优化图像。对于不透明度、最小和最大阈值、镜面、环境、Shineness 和伽玛, 存在用于优化图像的其他选项。</li>
		<li>通过使用裁剪和切片命令对模型中的剖面进行横断面, 以可视化全血细胞的内部内容。</li>
	</ol>
</li>
</ol>4. 基因和/或蛋白质分析的应用
<ol><li>继感染 IIV6 和李斯特菌后, 溶解细胞为随后的 qRT PCR 或西方印迹分析如前所述的21和以下制造商的指示。 注: 请参阅材料表, 用于 qRT PCR 的引物和用于西方印迹的抗体。</li>
	<li>用琼脂糖凝胶电泳分析 PCR 产物, 如前所述的22, 以确保放大产品的适当长度。</li>
</ol>

<ol>
	<li>Hoffmann, 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=The+immune+response+of+Drosophila.">The immune response of Drosophila.</a> Nature. 426 (6962), 33-38 (2003).</li><li>Regan, J. 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=Steroid+hormone+signaling+is+essential+to+regulate+innate+immune+cells+and+fight+bacterial+infection+in+Drosophila.">Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.</a> PLoS Pathog. 9 (10), 1003720 (2013).</li><li>Yano, T., 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=Autophagic+control+of+listeria+through+intracellular+innate+immune+recognition+in+Drosophila.">Autophagic control of listeria through intracellular innate immune recognition in Drosophila.</a> Nat Immunol. 9 (8), 908-916 (2008).</li><li>Bastos, R. G., Howard, Z. P., Hiroyasu, A., Goodman, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+Bacterial+Factors+Control+Susceptibility+of+Drosophila+melanogaster+to+Coxiella+burnetii+Infection.">Host and Bacterial Factors Control Susceptibility of Drosophila melanogaster to Coxiella burnetii Infection.</a> Infect Immun. 85 (7), (2017).</li><li>Kacsoh, B. Z., Schlenke, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+hemocyte+load+is+associated+with+increased+resistance+against+parasitoids+in+Drosophila+suzukii,+a+relative+of+D.+melanogaster.">High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster.</a> PLoS One. 7 (4), 34721 (2012).</li><li>Tsuzuki, S., 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=Switching+between+humoral+and+cellular+immune+responses+in+Drosophila.+is+guided+by+the+cytokine+GBP.">Switching between humoral and cellular immune responses in Drosophila. is guided by the cytokine GBP.</a> Nat Commun. 5, 4628 (2014).</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>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>Arefin, B., 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=Apoptosis+in+Hemocytes+Induces+a+Shift+in+Effector+Mechanisms+in+the+Drosophila.+Immune+System+and+Leads+to+a+Pro-Inflammatory+State.">Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila. Immune System and Leads to a Pro-Inflammatory State.</a> PLoS One. 10 (8), 0136593 (2015).</li><li>Rus, F., 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=Expression+pattern+of+Filamin-240+in+Drosophila+blood+cells.">Expression pattern of Filamin-240 in Drosophila blood cells.</a> Gene Expr Patterns. 6 (8), 928-934 (2006).</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=Hemese,+a+hemocyte-specific+transmembrane+protein,+affects+the+cellular+immune+response+in+Drosophila.">Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila.</a> P Natl Acad Sci USA. 100 (5), 2622-2627 (2003).</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> P Natl Acad Sci USA. 106 (12), 4805-4809 (2009).</li><li>Bretscher, A. 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=The+Nimrod+transmembrane+receptor+Eater+is+required+for+hemocyte+attachment+to+the+sessile+compartment+in+Drosophila+melanogaster.">The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster.</a> Biol Open. 4 (3), 355-363 (2015).</li><li>Zettervall, C. 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=A+directed+screen+for+genes+involved+in+Drosophila+blood+cell+activation.">A directed screen for genes involved in Drosophila blood cell activation.</a> P Natl Acad Sci USA. 101 (39), 14192-14197 (2004).</li><li>Flynt, A., Liu, N., Martin, R., Lai, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicing+of+viral+replication+intermediates+during+silencing+of+latent+Drosophila+viruses.">Dicing of viral replication intermediates during silencing of latent Drosophila viruses.</a> P Natl Acad Sci USA. 106 (13), 5270-5275 (2009).</li><li>Jovel, J., Schneemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+Drosophila+cells+persistently+infected+with+Flock+House+virus.">Molecular characterization of Drosophila cells persistently infected with Flock House virus.</a> Virology. 419 (1), 43-53 (2011).</li><li>Stoepler, T. M., Castillo, J. C., Lill, J. T., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+protocol+for+extracting+hemocytes+from+wild+caterpillars.">A simple protocol for extracting hemocytes from wild caterpillars.</a> J Vis Exp. (69), e4173 (2012).</li><li>Sampson, C. J., Williams, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+ex+vivo+incubation+of+Drosophila+primary+post-embryonic+haemocytes+for+real-time+analyses.">Protocol for ex vivo incubation of Drosophila primary post-embryonic haemocytes for real-time analyses.</a> Methods Mol Biol. 827, 359-367 (2012).</li><li>Nehme, N. T., 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=A+model+of+bacterial+intestinal+infections+in+Drosophila+melanogaster.">A model of bacterial intestinal infections in Drosophila melanogaster.</a> PLoS Pathog. 3 (11), 173 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+fruit+juice+egg+plates.">Drosophila fruit juice egg plates.</a> Cold Spring Harbor Protocols. (9), (2007).</li><li>Ahlers, L. R., Bastos, R. G., Hiroyasu, A., Goodman, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invertebrate+Iridescent+Virus+6,+a+DNA+Virus,+Stimulates+a+Mammalian+Innate+Immune+Response+through+RIG-I-Like+Receptors.">Invertebrate Iridescent Virus 6, a DNA Virus, Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors.</a> PLoS One. 11 (11), 0166088 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (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>Figliozzi, R. W., Chen, F., Chi, A., Hsia, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+inverse+Poisson+distribution+to+calculate+multiplicity+of+infection+and+viral+replication+by+a+high-throughput+fluorescent+imaging+system.">Using the inverse Poisson distribution to calculate multiplicity of infection and viral replication by a high-throughput fluorescent imaging system.</a> Virol Sin. 31 (2), 180-183 (2016).</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=Lamellocyte+differentiation+in+Drosophila.+larvae+parasitized+by+Leptopilina.">Lamellocyte differentiation in Drosophila. larvae parasitized by Leptopilina.</a> Dev Comp Immunol. 16 (2-3), 103-110 (1992).</li><li>Markus, R., Kurucz, E., Rus, F., Ando, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+wounding+is+a+minimal+and+sufficient+trigger+for+a+cellular+immune+response+in+Drosophila+melanogaster.">Sterile wounding is a minimal and sufficient trigger for a cellular immune response in Drosophila melanogaster.</a> Immunol Lett. 101 (1), 108-111 (2005).</li><li>McCormack, 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=Perforin-2+Protects+Host+Cells+and+Mice+by+Restricting+the+Vacuole+to+Cytosol+Transitioning+of+a+Bacterial+Pathogen.">Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen.</a> Infect Immun. 84 (4), 1083-1091 (2016).</li><li>Ozgen, 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=Construction+and+characterization+of+a+recombinant+invertebrate+iridovirus.">Construction and characterization of a recombinant invertebrate iridovirus.</a> Virus Res. 189, 286-292 (2014).</li><li>Jakob, N. J., Muller, K., Bahr, U., Darai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+first+complete+DNA+sequence+of+an+invertebrate+iridovirus:+coding+strategy+of+the+genome+of+Chilo+iridescent+virus.">Analysis of the first complete DNA sequence of an invertebrate iridovirus: coding strategy of the genome of Chilo iridescent virus.</a> Virology. 286 (1), 182-196 (2001).</li><li>Ghigo, E., Colombo, M. I., Heinzen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Coxiella+burnetii+parasitophorous+vacuole.">The Coxiella burnetii parasitophorous vacuole.</a> Adv Exp Med Biol. 984, 141-169 (2012).</li><li>Liu, F., 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=Drosophila+melanogaster+prophenoloxidases+respond+inconsistently+to+Cu2++and+have+different+activity+in+vitro.">Drosophila melanogaster prophenoloxidases respond inconsistently to Cu2+ and have different activity in vitro.</a> Dev Comp Immunol. 36 (3), 619-628 (2012).</li><li>De Gregorio, 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=An+immune-responsive+Serpin+regulates+the+melanization+cascade+in+Drosophila.">An immune-responsive Serpin regulates the melanization cascade in Drosophila.</a> Dev Cell. 3 (4), 581-592 (2002).</li><li>Kari, B., 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+raspberry+Gene+Is+Involved+in+the+Regulation+of+the+Cellular+Immune+Response+in+Drosophila+melanogaster.">The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster.</a> PLoS One. 11 (3), 0150910 (2016).</li><li>Wu, A. 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=Quantitative+assessment+of+single-cell+RNA-sequencing+methods.">Quantitative assessment of single-cell RNA-sequencing methods.</a> Nat Methods. 11 (1), 41-46 (2014).</li><li>Buettner, F., 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=Computational+analysis+of+cell-to-cell+heterogeneity+in+single-cell+RNA-sequencing+data+reveals+hidden+subpopulations+of+cells.">Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells.</a> Nat Biotechnol. 33 (2), 155-160 (2015).</li><li>Nevil, M., Bondra, E. R., Schulz, K. N., Kaplan, T., Harrison, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Binding+of+the+Conserved+Transcription+Factor+Grainy+Head+to+its+Target+Genes+Throughout+Drosophila+melanogaster+Development.">Stable Binding of the Conserved Transcription Factor Grainy Head to its Target Genes Throughout Drosophila melanogaster Development.</a> Genetics. 205 (2), 605-620 (2017).</li><li>Yang, C. P., 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=Transcriptomes+of+lineage-specific+Drosophila+neuroblasts+profiled+by+genetic+targeting+and+robotic+sorting.">Transcriptomes of lineage-specific Drosophila neuroblasts profiled by genetic targeting and robotic sorting.</a> Development. 143 (3), 411-421 (2016).</li><li>Jaitin, D. 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=Dissecting+Immune+Circuits+by+Linking+CRISPR-Pooled+Screens+with+Single-Cell+RNA-Seq.">Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq.</a> Cell. 167 (7), 1883-1896 (2016).</li><li>Karaiskos, N., 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+Drosophila+embryo+at+single-cell+transcriptome+resolution.">The Drosophila embryo at single-cell transcriptome resolution.</a> Science. 358 (6360), 194-199 (2017).</li></ol>

作者声明他们没有竞争的财政利益。

为了更好地了解宿主细胞是如何感染的, 重要的是要澄清细胞中病原体的定位, 尤其是在试验以前未经测试的病原体和细胞类型组合4时。在对感染后细胞反应级联进行研究时, 可能表明有生产性致病菌侵袭, 而影像学和细胞反应数据的结合对于证明病原菌侵袭和感染是必不可少的。虽然有报告显示病原体侵入宿主细胞的2D 图像往往表明有生产性感染, 但仍有一些问题可能存在于宿?...

果蝇是一种成熟的模型有机体, 用于研究先天免疫的1。在先天免疫反应中, 包囊在对病原体的挑战中起着重要的作用。包囊是关键的封装寄生虫, 以及有一个重要的功能, 通过吞噬行动在真菌, 病毒和细菌感染的病原体2,3。
为了更好地了解宿主对致病性微生物感染的先天免疫反应, 重要的是要想象病原体是如何侵入宿主细胞感染的。这种可视化有助于理解入侵的机制。这些数据连同病原体细胞内定位和细胞反应的细节, 可以提供关于宿主对感染的反应和微生物相互作用的细胞器的线索。因此, 采用显微成像技术进行3D 模型重建, 有助于确定宿主细胞病原体的准确定位。在本研究中, 我们可视化了贝柯克斯柯克斯 (柯克斯) 的入侵, "Q 发热" 的致病剂, 这是一种对人类和动物健康构成严重威胁的人畜共患病病毒, 成为主要的果蝇包囊. 最近, 据证明,果蝇易受生物安全级别2九英里第二阶段 (NMII) 克隆4菌株的C. 柯克斯的影响, 该菌株能够在果蝇4中复制, 表明果蝇可作为模型有机体研究C. 柯克斯发病机制。
以前的研究已经使用包囊检查宿主的先天免疫反应。包囊已用于形态学观察5,6,7, 形态分析2,8, 吞噬功能分析2,3, qRT PCR2, 9, 免疫沉淀 10, 11, 免疫荧光分析 10, 12, 染色 13, 免疫印迹法3,10, 11和免疫组化9,14。尽管果蝇S2 细胞也可用于各种体外实验, 但永生和潜在的病毒感染可能会改变其行为15,16。使用主细胞, 而不是永生化细胞系, 如 S2 细胞, 允许研究先天免疫功能的系统更具代表性的整个有机体。此外, 在提取之前, 包囊体内的感染允许细胞与其他宿主蛋白和组织进行交互, 这比在前体感染之前提取包囊更有利。许多不同的方法被用来在短时间内获得足够数量的包囊, 以保持包囊活着的8,17,18,19。
在本研究中, 我们提出了一种从果蝇3rd龄幼虫中提取包囊的方法, 用于致病性微生物感染C. 柯克斯,李斯特菌增生(李斯特菌), 或无脊椎动物彩虹病毒 6 (IIV6)。我们描述了体内和前体全血细胞感染的方法。体内-和前体感染的包囊通过共焦显微镜进行可视化, 用于构建C 柯克斯入侵的3D 模型。此外, 使用提取协议, 体外感染的包囊被用于基因和蛋白质表达测定. 具体地说, 为了检查感染的程度与 IIV6 和李斯特菌, 总 RNA 或蛋白质被分离从细胞为 qRT PCR 或西方印迹分析。结合起来, 该协议提供了从3个rd龄幼虫中快速收集大量包囊的方法, 并证明主要包囊感染了体内或前体内, 是一种适合微生物的平台。病原体感染研究及适用的下游分析, 如显微术、转录组学和蛋白质组学。

<table border="0" cellpadding="0" cellspacing="0" style="width:1128px;" width="1125">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:303px;">Schneider&#39;s Drosophila Medium&#160;</td>
			<td style="width:288px;">Thermo Fisher Scientific (Gibco)</td>
			<td style="width:145px;">21720024</td>
			<td style="width:179px;">1.1.1), 2.1.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:288px;">GE Healthcare Life Sciences&#160; (HyClone)</td>
			<td style="width:145px;">SH30070.03HI</td>
			<td style="width:179px;">1.1.1), 2.1.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Filter (0.22 &#181;L)</td>
			<td style="width:288px;">RESTEK</td>
			<td style="width:145px;">26158</td>
			<td style="width:179px;">1.1.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Strainer (100 &#181;m)</td>
			<td style="width:288px;">Greiner bio-one</td>
			<td style="width:145px;">542000</td>
			<td>1.2.1), 2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Stereo microscope</td>
			<td style="width:288px;">Amscope</td>
			<td style="width:145px;">SM-1BSZ-L6W</td>
			<td style="width:179px;">1.2), 2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Glass capillary</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">21-171-4</td>
			<td style="width:179px;">1.1), 1.2), 2)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Capillary puller</td>
			<td style="width:288px;">Narishige International USA, Inc.</td>
			<td style="width:145px;">PC-10</td>
			<td style="width:179px;">1.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mineral oil</td>
			<td style="width:288px;">Snow River Products</td>
			<td style="width:145px;"></td>
			<td style="width:179px;">1.1.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nanoinjector</td>
			<td style="width:288px;">Drummond Scientific Company</td>
			<td style="width:145px;">3-000-204</td>
			<td style="width:179px;">1.1), 1.2), 2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Forceps</td>
			<td style="width:288px;">VWR</td>
			<td style="width:145px;">82027-402</td>
			<td style="width:179px;">1.1.5), 1.2), 2), 3.1.7)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:303px;">CO2 delivery apparatus</td>
			<td style="width:288px;">Genesee Scientific</td>
			<td style="width:145px;">59-122BC</td>
			<td style="width:179px;">1.2), 2)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Trypan Blue</td>
			<td style="width:288px;">Thermo Fisher Scientific (Gibco)</td>
			<td style="width:145px;">15250061</td>
			<td style="width:179px;">1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemocytometer</td>
			<td style="width:288px;">Hausser Scientific</td>
			<td style="width:145px;">3100</td>
			<td style="width:179px;">1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">24 well plate</td>
			<td style="width:288px;">Greiner bio-one</td>
			<td style="width:145px;">662160</td>
			<td style="width:179px;">1.4), 2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Coxiella burnetii - mCherry</td>
			<td style="width:288px;">Dr. Heinzen, R.</td>
			<td style="width:145px;"></td>
			<td style="width:179px;">1.4), 2.2)</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:303px;">Drosophila fruit juice plates</td>
			<td style="width:288px;">Cold Spring Harbor Protocols</td>
			<td style="width:145px;"></td>
			<td>2.1) http://cshprotocols.cshlp.org/content/2007/9/pdb.rec11113.full</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Agar</td>
			<td style="width:288px;">Fisher Bioreagents</td>
			<td style="width:145px;">BP1423-500</td>
			<td style="width:179px;">2.1.1.1)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Methyl paraben</td>
			<td style="width:288px;">Amresco</td>
			<td style="width:145px;">0572-500G</td>
			<td style="width:179px;">2.1.1.2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Absolute ethanol</td>
			<td style="width:288px;">Fisher Bioreagents</td>
			<td style="width:145px;">BP2818-500</td>
			<td style="width:179px;">2.1.1.2)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Welch&#39;s 100% Grape juice frozen concentrate, 340 mL</td>
			<td style="width:288px;">Amazon</td>
			<td style="width:145px;">B0025UJVGM</td>
			<td style="width:179px;">2.1.1.3)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Petri dishes, 10 x 35 mm</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">08-757-100A</td>
			<td style="width:179px;">2.1.1.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope cover glass</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">12-545-80</td>
			<td style="width:179px;">1.4.4), 2.2.2)</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:303px;">Yeast, Bakers Dried Active</td>
			<td style="width:288px;">MP Biomedicals</td>
			<td style="width:145px;">0210140001</td>
			<td>2.1) Add 2 parts of water to 1 part of yeast (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tungsten needle</td>
			<td style="width:288px;">Fine Science Tools</td>
			<td style="width:145px;">10130-20</td>
			<td style="width:179px;">2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Holding forceps</td>
			<td style="width:288px;">VWR</td>
			<td style="width:145px;">HS8313</td>
			<td style="width:179px;">2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Paraformaldehyde</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">FLO4042-500</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triton X-100</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">BP151-500</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bovine Serum Albumin</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">BP9706-100</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">4&#39;,6-diamidino-2-phenylindole</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">62247</td>
			<td style="width:179px;">3.1.4)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Antifade mounting medium</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">P36930</td>
			<td style="width:179px;">3.1.6)</td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;width:303px;">Confocal microsope</td>
			<td style="width:288px;">Leica</td>
			<td style="width:145px;">TCS SP8-X White Light Confocal Laser Scanning Microscope</td>
			<td style="width:179px;">3.2)</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:303px;">3D imaging reconstruction software</td>
			<td style="width:288px;">Leica</td>
			<td style="width:145px;">LASX with 3D visualization module</td>
			<td style="width:179px;">3.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope slides</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">12-552-3</td>
			<td style="width:179px;">3.1.6)</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:303px;">Invertebrate iridescent virus 6 (IIV6)</td>
			<td style="width:288px;">Dr. Teixeria, L.</td>
			<td style="width:145px;"></td>
			<td>4) PLoS Biol, 6 (12), 2753-2763, doi: 10.1371/journal.pbio.1000002, (2008)</td>
		</tr>
		<tr height="141">
			<td height="141" style="height:141px;width:303px;">Listeria monocytogenes</td>
			<td style="width:288px;">ATCC</td>
			<td style="width:145px;">strain: 10403S</td>
			<td>4) Listeria monocytogenes strain 10403S (Bishop and Hinrichs, 1987) was grown in Difco Brain-heart infusion (BHI) broth (BD Biosciences) containing 50 &#181;g/ml streptomycin at 30 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">DNase I</td>
			<td style="width:288px;">Thermo Fisher Scientific(Invitrogen)</td>
			<td style="width:145px;">18068015</td>
			<td>gDNA degradation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">cDNA Synthesis Kit</td>
			<td style="width:288px;">Bio-Rad</td>
			<td style="width:145px;">1708891</td>
			<td>cDNA synthesis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">IIV6_193R_F</td>
			<td style="width:288px;">IDT</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- TCT TGT TTT CAG AAC CCC ATT -3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">IIV6_193R_R</td>
			<td style="width:288px;">IDT</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- CAC GAA GAA TGA CCA CAA GG -3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">RpII_qRTPCR_fwd</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- GAA GCG TTT CTC CAA ACG -AG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">RpII_qRTPCR_rev</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- TTG AGC GTA AGC ATC ACC -TG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SYBR Green qRT-PCR reagent</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">K0251, K0252, K0253</td>
			<td>qRT-PCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Real-Time PCR System</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">4351107, 7500 Software v2.0</td>
			<td>qRT-PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Listeria monocytogenes antibody</td>
			<td style="width:288px;">abcam</td>
			<td style="width:145px;">ab35132</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Actin antibody produced in rabbit</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;">A2066</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Rabbit IgG (H+L), HRP Conjugate</td>
			<td style="width:288px;">Promega</td>
			<td style="width:145px;">W4011</td>
			<td>Western blot</td>
		</tr>
	</tbody>
</table>

extraction of hemocytes from drosophila melanogaster larvae for microbial infection and analysis

在果蝇的致病性感染中,包囊在整个感染的免疫应答中起着重要作用。因此, 本议定书的目的是开发一种方法, 以可视化病原体入侵在特定的免疫室的苍蝇, 即包囊。使用此处提供的方法, 可从 200果蝇3rd龄幼虫到30分钟 (前体感染) 中获得多达 3 x 106实时包囊。或者, 通过注射 3rd龄幼虫, 然后全血细胞提取多达24小时后感染, 包囊可以感染体内。这些受感染的主要细胞是固定的, 染色, 并使用共聚焦显微镜成像。然后, 从图像中生成3D 表示, 最终显示病原体侵袭。此外, 还可以获得高质量的 qRT PCR RNA, 用于检测感染后的病原 mRNA, 并从这些细胞中提取足够的蛋白质进行印迹分析。结合, 我们提出了一个确定的方法, 对病原体入侵和证实感染的细菌和病毒病原体类型和有效的全血细胞提取方法获得足够的活包囊从果蝇用于前体和体内感染实验的幼虫。

要收集前体感染的实时包囊, 可从 200果蝇3rd龄幼虫中提取多达 3×106包囊。为了发展我们的方法, 我们尝试了许多不同的技术。个体幼虫解剖可达1.5 小时, 使用此方法18获得平均8000个细胞, 其中大部分在采集结束时未存活。接下来, 我们试图提取血淋巴, 其中包含包囊, 从20幼虫一次使用玻璃毛细管管19, 但毛细?...

Watch this Scientific Journal Video about Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis at JoVE.com

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

该方法展示了如何用三维 (3D) 模型来可视化病原体侵入昆虫细胞。包囊从果蝇幼虫感染病毒或细菌病原体, 无论是体内或体内。感染的包囊然后固定和染色的成像与共聚焦显微镜和随后的3D 细胞重建。

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 ...

extraction-hemocytes-from-drosophila-melanogaster-larvae-for

Research

JoVE Journal

Immunology and Infection

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

11.6K 次观看.  Washington State University. 该方法展示了如何用三维 (3D) 模型来可视化病原体侵入昆虫细胞。包囊从果蝇幼虫感染病毒或细菌病原体, 无论是体内或体内。感染的包囊然后固定和染色的成像与共聚焦显微镜和随后的3D 细胞重建。

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

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

科研

免疫与感染

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用"单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

全身细菌感染与免疫防御的表型<em&gt;果蝇</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

微生物菌落生物膜的石蜡嵌入和薄切片显微分析

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

弥散性念珠菌感染模型

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...