这项工作得到了由韩国政府 (MSIP) (NRF-2014K1A4A7A01074642) 和韩国国家研究基金会 (NRF) 个人科学家支持计划 (NRF-2013M3A9B5076486/NRF-2015R1D1A1A09057239) 资助的赠款。

1. 慢生产 注意: 根据国家卫生研究院 (NIH) 有关重组的研究指南, 对慢载体进行生产和操作。dna.用增强 YFP (SOD1WT-YFP 和 SOD1A4V-YFP) 标记的野生型和 A4V 突变体 SOD1 的质粒, 在金 et al 中描述。 11 两种基因融合产品均采用 PCR 引物对5和 #8242;-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3 和 #8242; 5 和 #8242;-TAGCGG CCGCTACTTGTACAGCTCGTCCATGCC-3 和 #8242; 插入 pTRIP 三角洲 U3 巨细胞病毒质粒 19 使用 XhoI 和 BsrGI 限制站点。在先前的实验中, HEK-293T 细胞的分裂率为1:4 至1:6 每两天。避免超过20通道和维持细胞在不到 60% 70-100 有助于确保良好的转染效率. 
<ol> <li> 在1天, 分裂 HEK-293T 细胞在 30-40% 汇合在 10 cm 直径组织培养板材 (3 x 10 6 细胞或盘) 在10毫升细胞培养基 (高 DMEM 与 10% FBS 和4毫米 l-谷氨酰胺) 为病毒生产. </li>
	<li> 保留10厘米直径组织培养板在 CO 2 孵化器 (37 和 #176; C, 5% CO 2 ) 过夜. </li>
	<li> 2 天, 准备一个含有10和 #181 的 DNA 转染溶液; g 慢载体 (pTRIP U3 CMV SOD1WT-YFP 或-SOD1A4V-YFP) 连同慢包装质粒 (5 和 #181; g pVSVg; 10 和 #181; g pCMV-dR8.71) ( 图 2A ) 20 . 添加125和 #181; 1 M CaCl 2 , 并将音量调整为500和 #181; 我使用蒸馏水。轻轻地加500和 #181; 0.05 米 HEPES 到混合物中, 室温下孵育10分钟. </li>
	<li> 用10毫升5ml 新鲜培养基取代 HEK-293T 细胞培养基与1毫升 DNA 转染溶液混合, 并在夜间孵育37和 #176; C, 5% CO 2 . </li>
	<li> 在3天, 用新鲜培养基取代细胞上清液. </li>
	<li> 在4天, 在15毫升的管中收获上清液, 并在 500 x g 离心5分钟, 以去除死细胞和碎片。通过0.45 和 #181 进一步净化上清液; m 过滤器使用大60毫升注射器。立即将一次性等分 (300 和 #181; L), 并存储在-80 和 #176; C。要保持最大的产品活动, 请避免冻结解冻周期. </li>
</ol> 2。慢转导 <ol> <li> 将单元格线拆分到60% 汇合处 (HEK-293 细胞和 SH-SY5Y; 5 x 10 5 单元格和 U2OS; 每井 1 x 10 个, 在 5-良好组织培养板块中6毫升的 DMEM 培养基中补充10% FBS. </li>
	<li> 将6个组织培养板保持在37和 #176; C 孵化器在 5% CO 2 过夜. </li>
	<li> 将冻结的慢从-80 和 #176 中取出; 在每次使用前将冰分冷冻并解冻; 不要冻结. </li>
	<li> 当病毒正在解冻时, 将含有血清的细胞培养基加热, 以符合细胞系的利益。一旦病毒完全解冻, 准备一个稀释 (1:3, 1:10, 一点半, 1:100) 在 DMEM 在一个新鲜的1.5 毫升离心管的范围. </li>
	<li> 将试管中的音量调到1毫升, 并降低血清介质. </li>
	<li> 添加2和 #181; 凝聚 (在4和 #181 的股票; g/和 #181; l) 到1毫升的病毒/媒体。移混合均匀, 并加入1毫升的混合物到细胞。用病毒孵育细胞 24 h. </li>
	<li> 删除病毒介质并替换为正常的 DMEM 介质, 并辅以 10% FBS。保持37和 #176 的单元格; C, 5% CO 2 . </li>
	<li> 监视单元格的增长并每两天更改一次区域性媒体。在汇合处, 将6井道扩展成直径10厘米的组织培养皿. </li>
	<li> 一旦单元格已得到充分扩展, 种子 1.5 x 10 4 每井50和 #181 的细胞; DMEM 培养基上的 384-试板, 通过显微镜检查 YFP 标记蛋白的表达水平。如果 YFP 标记的蛋白质细胞表达显示一个信噪比和 #8805; 3, 为相应的细胞线准备细胞储存. </li>
</ol> 3。蛋白酶体抑制剂的时间依赖性对蛋白质聚集的影响 注意: 使用自动显微镜执行图像采集 (请参阅 材料 )。
<ol> <li> 免除0.25 和 #181; 在 384-井平底板上, 亚砜或蛋白酶体抑制剂 L 的溶解在 100% ALLN, 2 毫米) 上. </li>
	<li> 手动播种 1.5 x 10 4 每个井在50和 #181 中的单元格; DMEM 介质在同一检测板上。蛋白酶体抑制剂 (ALLN) 最终浓度应为10和 #181; m. </li>
	<li> 将显微镜系统环境控制单元设置为37和 #176; C 和 5% CO 2 。实验设置的屏幕截图如 图 3 所示. </li>
	<li> 在广域荧光模式下操作显微镜, 使用以下设置的软件: 钻20X 目标, non-confocal 模式, 4 字段/好, 捕获间隔为一小时。每次运行结束时, 捕获的图像都会自动上载到服务器. </li>
</ol> 4。活细胞中 YFP 表达的时间推移成像和图像分析 (单通道) 注意: 以下步骤描述了软件的应用 ( 如 , 哥伦布)。
<ol> <li> 选择适当的算法来分割主要对象 (胞和聚合)。如果需要, 请调整背景阈值和对比度参数 ( 图 4 ). </li>
	<li> 使用和 #39 计数单元格; 查找单元格和 #39; YFP 强度通道的单个阈值为0.1。使用 and #39 确定聚合; 查找点和 #39; 具有相对 YFP 点强度和 #62 的算法; 0.2 和分裂系数为 1.0. </li>
</ol> 5。蛋白酶体抑制剂对活细胞蛋白质聚集的剂量响应效应 (两个通道) 注意: 使用自动显微镜进行图像采集。根据预期的 IC 50 值确定蛋白酶体抑制剂 (ALLN、Epoxomicin 和 MG132) 的浓度范围, 以确保最佳的曲线拟合. 
<ol> <li> 用标准的1:3 稀释系列准备 384-井储存聚丙烯板上的化合物的系列稀释。一定要把复合稀释好, 以确保化合物的浓度是准确的. </li>
	<li> 免除0.25 和 #181; 在空扁底黑384试板上的蛋白酶体抑制剂 L。我们使用的液体处理程序配备了384毛细管头能够转移体积. </li>
	<li> 种子 1.5 x 10 4 每个井在50和 #181 中的单元格; DMEM 介质在检测板上的 L。在37和 #176 上孵育检测板; C 和 5% CO 2 , 用于 24 h. </li>
	<li> 添加10和 #181; DMEM 介质 (5ml 37 和 #176; C) 与染色液 (赫斯特 33342, 10 毫克/毫升), 室温孵育10分钟. </li>
	<li> 启动百万分之一处理操作软件。实验设置的屏幕截图如 图 6 所示。选择 #34; 配置和 #34; 选项卡并选择20X 和 #160; 目标和正确的板型。确保和 #34; 衣领和 #34; 在目标上设置正确的值, 以便在不同的板型中进行适当的聚焦. </li>
	<li> 选择 #34; 显微镜和 #34; 标签. 定义曝光1作为 YFP (488 laser) 和曝光2作为赫斯特 (405 laser)。激活两个曝光的过滤器, 并将曝光1分配给照相机1和曝光2到照相机2。将曝光时间设为800毫秒, 曝光率为 1, 40 毫秒, 曝光率为 2. </li>
	<li> 选择曝光1。将焦点高度设置为0和 #181; m. 选择和 #34; 焦点和 #34;。一旦聚焦, 曝光摄像头1。调整焦点高度以优化曝光平面, 然后单击并 #34; 采取高度和 #34;。改变曝光时间和激光功率, 使最大像素强度为3000。保存曝光参数。重复曝光 2. </li>
	<li> 选择和 #34; 实验定义和 #34; 选项卡. 创建布局和 sublayout。拖放相关的布局、曝光、参考图像、skewcrop 文件和 sublayout。保存实验. </li>
	<li> 选择和 #34; 自动实验和 #34; tab 并获取图像。每次运行结束时, 捕获的图像都会自动上载到服务器. </li>
</ol> 6。两个通道图像的图像分析 <ol> <li> 选择软件算法来分割主对象 (核、胞和聚合) ( 图 7 )。
	<li> 通过对分段对象进行目视检查, 选择精确细分原子核的方法。赫斯特通道 1 (Ch1) 中的染色对象将用于确定单元格的数量。在通道 2 (Ch2) 的突变 SOD1-A4V 的 YFP 染色将用于确定总量. </li>
	<li> 使用 and #39 计数单元格; 查找核和 #39; 算法为赫斯特染色区域和 #62; 20 和 #181; m 2 , 其分割因子为 7.0, 单个阈值为 0.40, 对比度和 #62; 0.10. </li>
	<li> 创建数据表并确定 EC 50 使用 and #39 的每个化合物; 非线性回归和 #39; 绘图软件中的等式. </li>
</ol>

<ol>
	<li>Schulte, J., Sepp, K. J., Wu, C., Hong, P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-content+chemical+and+rnai+screens+for+suppressors+of+neurotoxicity+in+a+huntington's+disease+model.">High-content chemical and rnai screens for suppressors of neurotoxicity in a huntington's disease model.</a> PLoS ONE. 6 (8), e23841 (2011).</li><li>Honarnejad, K., 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=Development+and+implementation+of+a+high-throughput+compound+screening+assay+for+targeting+disrupted+ER+calcium+homeostasis+in+Alzheimer's+disease.">Development and implementation of a high-throughput compound screening assay for targeting disrupted ER calcium homeostasis in Alzheimer's disease.</a> PLoS ONE. 8 (11), 1-12 (2013).</li><li>Burkhardt, M. 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=A+cellular+model+for+sporadic+ALS+using+patient-derived+induced+pluripotent+stem+cells.">A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells.</a> Mol Cell Neurosci. 56, 355-364 (2013).</li><li>Mattiazzi, U., 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=High-Content+Screening+for+Quantitative+Cell+Biology.">High-Content Screening for Quantitative Cell Biology.</a> Trends Cell Biol. 26 (8), 598-611 (2016).</li><li>Kato, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyotrophic+lateral+sclerosis+models+and+human+neuropathology:+Similarities+and+differences.">Amyotrophic lateral sclerosis models and human neuropathology: Similarities and differences.</a> Acta Neuropathologica. 115 (1), 97-114 (2008).</li><li>Strong, M. J., Kesavapany, S., Pant, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathobiology+of+amyotrophic+lateral+sclerosis:+a+proteinopathy?.">The pathobiology of amyotrophic lateral sclerosis: a proteinopathy?.</a> J neuropathol exp neurol. 64 (8), 649-664 (2005).</li><li>Cleveland, D. W., Rothstein, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Charcot+to+Lou+Gehrig:+deciphering+selective+motor+neuron+death+in+ALS.">From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS.</a> Nat rev Neurosci. 2 (11), 806-819 (2001).</li><li>Bruijn, L. I., Miller, T. M., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+Mechanisms+Involved+in+Motor+Neuron+Degeneration+in+Als.">Unraveling the Mechanisms Involved in Motor Neuron Degeneration in Als.</a> Annu Rev Neurosci. 27 (1), 723-749 (2004).</li><li>Vassall, K. 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=Decreased+stability+and+increased+formation+of+soluble+aggregates+by+immature+superoxide+dismutase+do+not+account+for+disease+severity+in+ALS.">Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS.</a> Proc Natl Acad Sci USA. 108 (6), 2210-2215 (2011).</li><li>Hwang, Y. M., 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=Nonamyloid+aggregates+arising+from+mature+copper/zinc+superoxide+dismutases+resemble+those+observed+in+amyotrophic+lateral+sclerosis.">Nonamyloid aggregates arising from mature copper/zinc superoxide dismutases resemble those observed in amyotrophic lateral sclerosis.</a> J Biol Chem. 285 (53), 41701-41711 (2010).</li><li>Kim, 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=Dimerization,+Oligomerization,+and+Aggregation+of+Human+Amyotrophic+Lateral+Sclerosis+Copper/Zinc+Superoxide+Dismutase+1+Protein+Mutant+Forms+in+Live+Cells.">Dimerization, Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells.</a> J Biol Chem. 289 (21), 15094-15103 (2014).</li><li>Shaw, B. 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=Detergent-insoluble+aggregates+associated+with+amyotrophic+lateral+sclerosis+in+transgenic+mice+contain+primarily+full-length,+unmodified+superoxide+dismutase-1.">Detergent-insoluble aggregates associated with amyotrophic lateral sclerosis in transgenic mice contain primarily full-length, unmodified superoxide dismutase-1.</a> J Biol Chem. 283 (13), 8340-8350 (2008).</li><li>Banci, L., 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=SOD1+and+amyotrophic+lateral+sclerosis:+Mutations+and+oligomerization.">SOD1 and amyotrophic lateral sclerosis: Mutations and oligomerization.</a> PLoS ONE. 3 (2), 1-8 (2008).</li><li>Johnston, J. a., Dalton, M. J., Gurney, M. E., Kopito, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+high+molecular+weight+complexes+of+mutant+Cu,+Zn-superoxide+dismutase+in+a+mouse+model+for+familial+amyotrophic+lateral+sclerosis.">Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis.</a> Proc Natl Acad Sci. 97 (23), 12571-12576 (2000).</li><li>Niwa, J. I., 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=Disulfide+bond+mediates+aggregation,+toxicity,+and+ubiquitylation+of+familial+amyotrophic+lateral+sclerosis-linked+mutant+SOD1.">Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1.</a> J Biol Chem. 282 (38), 28087-28095 (2007).</li><li>Juneja, 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=Prognosis+in+familial+amyotrophic+lateral+sclerosis:+progression+and+survival+in+patients+with+glu100gly+and+ala4val+mutations+in+Cu,Zn+superoxide+dismutase.">Prognosis in familial amyotrophic lateral sclerosis: progression and survival in patients with glu100gly and ala4val mutations in Cu,Zn superoxide dismutase.</a> Neurology. 48 (1), 55-57 (1997).</li><li>Kaur, S. J., McKeown, S. R., Rashid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+SOD1+mediated+pathogenesis+of+Amyotrophic+Lateral+Sclerosis.">Mutant SOD1 mediated pathogenesis of Amyotrophic Lateral Sclerosis.</a> Gene. 577 (2), 109-118 (2016).</li><li>de Bruyns, A., Geiling, B., Dankort, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+Modular+Lentiviral+Vectors+for+Effective+Gene+Expression+and+Knockdown.">Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.</a> Methods Mol Biol. 1448, 3-21 (2016).</li><li>Sirven, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Transgene+Expression+in+Cord+Blood+CD34+-Derived+Hematopoietic+Cells,+Including+Developing+T+Cells+and+NOD/SCID+Mouse+Repopulating+Cells,+Following+Transduction+with+Modified+TRIP+Lentiviral+Vectors.">Enhanced Transgene Expression in Cord Blood CD34+-Derived Hematopoietic Cells, Including Developing T Cells and NOD/SCID Mouse Repopulating Cells, Following Transduction with Modified TRIP Lentiviral Vectors.</a> Mol Ther. 3 (4), 438-448 (2001).</li><li>Wu, C., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-titre+retroviral+vector+system+for+efficient+gene+delivery+into+human+and+mouse+cells+of+haematopoietic+and+lymphocytic+lineages.">High-titre retroviral vector system for efficient gene delivery into human and mouse cells of haematopoietic and lymphocytic lineages.</a> J gen virol. 91 (Pt 8), 1909-1918 (2010).</li><li>Irshad, H., Veillard, A., Roux, L., Racoceanu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+nuclei+detection,+segmentation,+and+classification+in+digital+histopathology:+A+review-current+status+and+future+potential.">Methods for nuclei detection, segmentation, and classification in digital histopathology: A review-current status and future potential.</a> IEEE Rev Biomed Eng. 7, 97-114 (2014).</li><li>Purschke, M., Rubio, N., Held, K. D., Redmond, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxicity+of+Hoechst+33342+in+time-lapse+fluorescence+microscopy.">Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy.</a> Photochem Photobiol Sci. 9 (12), 1634-1639 (2010).</li><li>Jaskova, K., Pavlovicova, M., Jurkovicova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transporters+and+their+role+in+the+development+of+neuronal+disease+and+neuronal+damage.">Calcium transporters and their role in the development of neuronal disease and neuronal damage.</a> Gen physiol biophys. 31 (4), 375-382 (2012).</li><li>Kieran, 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=Treatment+with+arimoclomol,+a+coinducer+of+heat+shock+proteins,+delays+disease+progression+in+ALS+mice.">Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice.</a> Nat Med. 10 (4), 402-405 (2004).</li></ol>

作者没有什么可透露的。

有两种主要的方法来生成稳定的细胞系。第一次需要几个星期, 需要对基因组整合的质粒 DNA 载体进行瞬时转染和抗性选择。第二个需要几个小时的时间, 通过使用慢, 使这一协议的有效表达的目标蛋白在多个细胞系有限的努力。行程-CMV 矢量19是一个持续使用的载体, 具有保守的转导效率和稳定的转基因表达。令我们吃惊的是, 生成的三单元格线显示的是突变 SOD1 的可见聚合, 而不是基于前面描述的11的瞬态表达式的实验协议。在相对较短的时间内产生更高的蛋白质表达, 瞬态表达可能有利于蛋白质的聚集。在瞬变转染过程中, ubiquitin–proteasome 系统会使大多数蛋白质降解, 从而达到饱和点, 从而充分利用其降解聚合易感蛋白的能力。然而, 我们认为, 稳定的线更有可能重现的生理状况, 其中蛋白质表达在一个相对恒定的水平, 匹配消除错误蛋白通过蛋白酶体, autophagosome-溶,胞.虽然主要事件导致 ALS 仍然未知, 老化与泛素蛋白酶体系统活性降低有关, 是一个重要的风险因素23。正如我们的细胞分析所示, 蛋白酶体抑制促进了错误蛋白 (SOD1 A4V) 的聚集。因此, 这种分析创造了新的机会, 筛选小分子激活的自噬信号通路或陪护网络。的确, 这一途径的伴侣感应似乎是有希望的, 如报告所示, 从al.的研究表明, co-inducer 的治疗, 热休克反应被发现有治疗效益, 延长 SODG93A 的寿命鼠标24。
重要的是要提到, 有几个陷阱需要考虑之前, 使用这种化验。与分析优化协议一样, 我们发现, 1% 或以上的亚甲基亚砜浓度增加了测定 YFP 的平均强度。因此, 必须将亚甲基亚砜的浓度降低到 0.5%, 以避免这种人工制品。此外, 选择的镜头放大率和细胞密度的检测板是重要的考虑, 以优化的鲁棒性分析。
在我们的研究中, 我们已经表明, 使用慢系统, SOD1A4V-YFP HEK-293 细胞线很适合监测突变体 SOD1 A4V 聚合形成后, 诱导蛋白酶抑制剂。错误蛋白和聚集性的研究对于了解 proteinopathies 的机制是至关重要的。尽管在过去的几十年中, 分析蛋白质聚集的研究工具已经扩大, 但需要有效的方法来检测和量化聚合, 以筛选禁止聚合形成的分子。这项工作提供了一个详细的协议, 分析发展和研究蛋白质聚集。希望在 HCS、HCA 和使用化学或 CRISPR/siRNA 图书馆的支持下, 这项工作应该开辟新的疗法或新奇的目标发现途径。

蛋白质聚集是一种生物过程, 错误蛋白可以在神经退行性疾病 (淀粉样变性) 中起到致病剂的作用。在理解细胞功能障碍中聚合体的作用, 以及在促进发现影响病理发病的新因素方面, 蛋白质聚集性是必不可少的。活体细胞荧光标记蛋白的可视化是一种强有力的方法, 可以帮助开发适用于高含量筛选 (HCS)1、2、3、4的测试。
肌萎缩侧索硬化 (als) 被认为是一种 proteopathic 的疾病, 由存在的错误蛋白的倾向聚集和积累在运动神经元的家族 als (fALS) 和零星 als (销售)5,6.20% fALS 病例的一个子集与胞浆抗氧化酶铜锌超氧化物歧化因子 1 (SOD1)7,8的基因编码中的显性突变相关。提出了这一基因衍生性功能障碍的几个潜在原因, 包括 SOD1 变种的结构和功能的变化, 如异常稳定性, 增加的展开率, 和倾向于聚合9, 10。值得注意的是, 由 ALS 连接的 SOD1 变种和野生型 (重量) SOD1 所共享的唯一经过验证和潜在毒性的属性是形成条块分割的蛋白质聚合体或蛋白质夹杂物的倾向11,12.错误突变 SOD1, 是持续 polyubiquitinated 和退化的泛素-蛋白酶体系统。因此, 对蛋白酶体活性的抑制导致了突变体 SOD1 聚集13,14, 形成了由可溶性组分组成的无定形结构, 可与可溶性突变体 SOD1 交换。在胞15中。特别是, SOD1 突变 A4V (丙氨酸在密码子4改为缬氨酸) 是最常见的 ALS 引起的突变, 并导致快速神经的平均生存时间少于2年后疾病发作16。生化, SOD1 A4V 有增加的倾向 monomerize, 聚合, 并形成淀粉样孔隙;它的孔状聚合体类似于其他与疾病相关的突变体的淀粉样孔, 如α-核和β-淀粉样蛋白17。为了研究 SOD1-aggregate 积累的动力学规律, 对可溶性和不溶性 SOD1 聚合形式的监测方法有待发展。
我们以前已经显示, 使用活细胞成像和 HEK-293 细胞瞬时转染荧光蛋白标记 SOD1, ALS 相关的突变损害 SOD1 二和聚集11。虽然瞬态表达系统可以提供有关短期基因过度过度的生物学结果的有用信息, 但提供所需基因的稳定整合的方法可能更适合于检测的发展。因此, 慢载体提供了赋予哺乳动物细胞长期和调控基因表达的能力18。在这项研究中, 我们的重点是生成稳定细胞系转与重组慢轴承和突变 SOD1 标记的黄色荧光蛋白 (YFP)。使用活细胞成像显微镜和自动定量的 SOD1 聚集, 我们触发和量化 SOD1 聚集事件时抑制蛋白酶体。

<table>
	<tbody>
		<tr>
			<td>ALLN (C20H37N3O4)</td>
			<td>Millipore</td>
			<td>208719</td>
			<td></td>
		</tr>
		<tr>
			<td>MG132 (C26H41N3O5)</td>
			<td>Sigma-Aldrich</td>
			<td>C2211</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxomicin (C28H50N4O7)</td>
			<td>Sigma-Aldrich</td>
			<td>E3652</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H-3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera</td>
			<td>Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera EvoShell software</td>
			<td>Perkin Elmer</td>
			<td>Ver 1.8.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Operetta</td>
			<td>Perkin Elmer</td>
			<td>OPRT1288</td>
			<td></td>
		</tr>
		<tr>
			<td>Harmony Imaging software</td>
			<td>Perkin Elmer</td>
			<td>Ver 3.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbus Image analysis software</td>
			<td>Perkin Elmer</td>
			<td>Ver 2.3.2</td>
			<td></td>
		</tr>
		<tr>
			<td>CyBi Hummingwell liquid handling</td>
			<td>CyBio AG</td>
			<td>OL 3387 3 0110</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay development for high content quantification of sod1 mutant protein aggregate formation in living cells

肌萎缩侧索硬化症 (ALS) 是一种致命的神经退行性疾病, 可导致遗传突变的基因编码铜锌超氧化物歧化酶 1 (SOD1)。SOD1 的结构不稳定性和家族性 als 患者的 SOD1-positive 包裹体的检测支持错误和/或聚合 SOD1 在 als 病理中的潜在因果作用。在这项研究中, 我们描述了一个细胞分析的发展, 旨在量化的动态 SOD1 聚集在活体细胞的高含量的筛选方法。利用慢向量, 我们生成了表达野生型和突变 A4V SOD1 标记的稳定细胞系, 黄色荧光蛋白, 发现这两种蛋白质都表达了胞没有任何聚集的迹象。有趣的是, 只有 SOD1 A4V 稳定表达在 HEK-293, 但不是在 U2OS 或 SH-SY5Y 细胞线, 形成了聚集后, 蛋白酶体抑制剂治疗。我们表明, 这是可能的量化聚合的基础上, 剂量反应分析的各种蛋白酶体抑制剂, 并跟踪集料形成动力学的延时显微镜。我们的方法引入了定量的可能性, ALS 突变的作用, SOD1 在聚合形成和筛选的小分子, 防止 SOD1 A4V 聚集。

使用慢生成稳定的单元格线:在图 1中说明了监测 SOD1 蛋白聚合体的总体策略。在第一步, 我们生成了一个慢表达载体, SOD1 稳定基因传递到细胞系 (步骤 1)。制备了两个慢矢量编码 YFP 标签 SOD1 和 SOD1 A4V (SOD1WT-YFP 和 SOD1A4V-YFP 分别) 与包装和包络质粒 (图 2A)。在慢转导 (步骤 2) 测试的细胞线 (HEK-293, U2OS 和 SH-SY5Y) 保持健康, 显示高表达水平 (图 2B), 和长期 YFP 标签, 无论 SOD1 的形式。有趣的是, 无论是重量或突变 SOD1 表达的三细胞线, 显示可见的聚集, 相比之下, 瞬态表达式蜂窝模型以前测试11。
验证 SOD1 聚合的稳定单元格线并研究其动态:在错误 SOD1 突变体细胞积累的基础上, 采用蛋白酶抑制剂 ALLN (也称为蛋白酶 I 和 II 抑制剂, 以触发 SOD1 聚集形成;分别为190和 220 nM), 以前使用瞬态 SOD1 表达式蜂窝模型11进行了验证。用 HEK-293、U2OS 和 SH-SY5Y 细胞系转与 SOD1WT-YFP 和 SOD1A4V-YFP SOD1 聚集进行了检查。用蛋白酶体抑制剂 ALLN (10 µM) 治疗24小时, 强烈促进了 HEK-293 细胞中 SOD1A4V-YFP 聚集物的积累 (图 2C)。然而, 在 SOD1WT-YFP 转细胞系中未发现聚合, U2OS 和 SH-SY5Y 细胞系 SOD1A4V-YFP 转 SOD1WT-YFP 和 SOD1A4V-YFP (图 2C) 中发现了极低的聚合水平。基于这些观察, 我们使用时移显微镜研究蛋白酶体抑制诱导的 SOD1A4V-YFP 聚集形成的动态 (步骤 3,图 3), 并利用单 YFP 荧光来量化 SOD1A4V-YFP 聚合用于检测活细胞中的表达单元和聚合体的通道 (步骤 4,图 4)。细胞在存在和无 ALLN 的情况下播种, 并监测50小时 (图 5A)。在我们的实验条件下, 我们发现 ALLN 诱导的聚合体形成在24小时内到达一个高原, 在接下来的12小时内保持稳定。我们表明, 28 小时后细胞密度明显下降, 由于 ALLN 的细胞毒作用, 而细胞数量增加在亚砜治疗阴性实验。
表征小分子 EC50用于使用 SOD1 单元模型的聚合生成:表达 SOD1A4V-YFP 的细胞以剂量依赖性的方式处理, 小分子蛋白酶体抑制剂。我们使用核标记来标记 SOD1A4V-YFP 细胞系, 这有利于在胞舱内进行聚合检测。事实上, 由于每个细胞都含有一个细胞核, 通过分割细胞核并在细胞的分水岭分割中使用它们作为种子, 细胞质分割被简化为21。我们使用赫斯特染色, 这是已知没有毒性时使用的短期和低浓度的22, 在以前的时间推移成像实验没有使用的条件。经过24小时的细胞培养在存在的蛋白酶体抑制剂, SOD1A4V-YFP 细胞染色赫斯特溶液 (10 µg/毫升) 10 分钟。接下来, 我们使用 20X 0.4 NA 目标 (步骤 5,图 6) 获取赫斯特和 YFP 的荧光图像。利用图像分析软件 (步骤 6,图 7) 对细胞细胞核和 SOD1A4V-YFP 分布的图像分析算法进行了分析。在背景上显示适当荧光强度的赫斯特染色物体和细胞核的形态大小特征 (宽度、长度和面积) 被识别并按图像分割进行分类。从赫斯特染色中提取的核面罩用于跟踪细胞胞浆区内的近 SOD1A4V-YFP 斑分布, 并确定团聚体的存在或缺失。在核和斑点检测的基础上, 计算了集料与细胞的比值。为了确定我们的检测的灵敏度, 我们的下一个量化聚合的 SOD1A4V-YFP HEK-293 细胞处理三不同的蛋白酶体抑制剂 (ALLN, MG132, 和 epoxomicin) 的剂量浓度的方式, 使拟合的量化数据和我们分别计算了 EC50值6.53 ±1.17、0.17 ±0.06 和0.03 ±0.03 µM 用于 ALLN、MG132 和 epoxomicin (图 8B)。所有三蛋白酶体抑制剂的相对毒性是量化的, 通过测量在标记为赫斯特染料的井中残存的黏附细胞的数量。我们发现, 总的细胞数趋于减少, 以响应复合效应, 证明了类似的 EC50值6.58 ± 1.06, 0.19 ± 0.03, 0.05 ±0.01 µM 为 ALLN, MG132 和 epoxomicin, 分别。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56425/56425fig1.jpg"> 
图1。蛋白质聚集的细胞生成和高含量分析 (HCA) 的工作流程和时间线.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56425/56425fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56425/56425fig2.jpg"> 
图2。SOD1 和 A4V 稳定线生成。 
(A)用于野生类型和突变 SOD1 A4V 慢生成的慢和包装载体 (包装和封套质粒) 的原理图。(B)选定的共聚焦图像的三细胞 (HEK-293, U2OS, 和 SH-SY5Y) 转慢野生类型 SOD1 (重量) 和突变 SOD1 (A4V)。(C)用蛋白酶体抑制剂 ALLN (10µM) 治疗24小时的三稳定细胞的代表性图像。SOD1A4V-YFP 聚集物被发现是大量存在 (红色箭头) 在 HEK-293, 但稀疏存在于 U2OS 或 SH-SY5Y 细胞。使用20X 目标获取图像。缩放栏 = 50 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56425/56425fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56425/56425fig3.jpg"> 
<str></str>图3。实验设置的屏幕快照使用了受控环境条件的显微镜系统 (37 ° c 和 5% CO2)。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56425/56425fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56425/56425fig4.jpg"> 
图4。使用 YFP 通道检测单元和聚合体的图像分析所需的四步骤。 (1) 从图像数据存储和分析系统中导入图像。(2) 为计数单元选择 "查找单元格"。(3) 选择 "查找点" 以确定合计。(4) 根据每个算法定义结果。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56425/56425fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56425/56425fig5.jpg"> 
图5。蛋白酶体抑制导致 HEK-293 细胞中 SOD1A4V-YFP 聚集的积累。 
(A)用10µM ALLN 处理的 SOD1A4V-YFP HEK-293 细胞的共聚焦图像。(B)对 ALLN (10µM) 和细胞计数引起的集料形成的定量分析。图像获得使用钻20X 目标每60分钟规模酒吧 = 50 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56425/56425fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56425/56425fig6.jpg"> 
图6。屏幕拍摄的实验设置为两个通道采集 (YFP 和赫斯特).<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56425/56425fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56425/56425fig7.jpg"> 
图7。SOD1 聚合体和 YFP 和赫斯特标记的细胞的图像分析所需的四步骤。 (1) 从图像数据存储和分析系统中输入图像。(2) 选择 "寻找细胞核" 来计数细胞。(3) 选择 "查找点" 以确定合计。(4) 根据每个算法定义结果。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56425/56425fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56425/56425fig8.jpg"> 
图8。SOD1 A4V 聚集和剂量依赖性蛋白酶体抑制剂浓度的定量分析。 
(A)在这里, 我们使用哥伦布图像分析系统对斑点和细胞计数的图像和量化分析方法进行了介绍。对应于赫斯特 (Ch1) 和 YFP (Ch2) 的两个荧光通道的图像依次获得。用图像分割法对赫斯特染色物体 (左) 进行识别。核掩模 (中心) 派生自 "发现核" 算法应用于计数的细胞数, 其次是斑点掩码 (右) "发现点" 算法用于检测集料。(B)对蛋白酶体抑制剂对突变 SOD1 A4V 聚合影响的剂量反应研究, 该方法显示可变 EC50排名从6.53 µM, ALLN, 到0.17 µM 为 MG132, 0.03 µM 为 Expoxomicin。在 EC50浓度下, SOD1 A4V 处理的蛋白酶体的选择共聚焦图像。缩放栏 = 50 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56425/56425fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells at JoVE.com

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

我们描述一种方法来量化错误蛋白质的聚集。我们的协议细节慢诱导稳定细胞线生成, 自动共焦成像, 和图像分析的蛋白质集料。作为一个说明性应用, 我们研究了小分子在促进 SOD1 聚集的时间和剂量依赖性的方式的影响。

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

assay-development-for-high-content-quantification-sod1-mutant-protein

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.1K 次观看.  Institut Pasteur Korea. 我们描述一种方法来量化错误蛋白质的聚集。我们的协议细节慢诱导稳定细胞线生成, 自动共焦成像, 和图像分析的蛋白质集料。作为一个说明性应用, 我们研究了小分子在促进 SOD1 聚集的时间和剂量依赖性的方式的影响。

视频: Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that 
initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The 
recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.
Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the 
photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by 
the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all-
trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is 
then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).
Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer 
segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. 
Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. 
Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single 
photoreceptor cells17-19.

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

生活荧光成像的隔离脊椎动物的感光细胞的制备

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

科研

神经科学

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

定量细胞迁移检测方法的鼠肠神经前体细胞

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

高含量的成像检测方法的肉毒毒素抑制剂的鉴定

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

简单的检测方法的社交回避的量化<em&gt;果蝇</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

果蝇岛试验中运动行为的高通量分析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

温度梯度法测定果蝇幼虫的热偏好

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

定量自动放射法测定维沃脑蛋白合成区域速率

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...