Name: supervised machine learning for semi-quantification of extracellular dna in glomerulonephritis
Uploaded: 2020-06-18T14:00:00
Description: Watch this Scientific Journal Video about Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis at JoVE.com

我们承认莫纳什微成像用于使用尼康C1直立共聚焦激光扫描显微镜和莫纳什组织学平台处理肾脏组织。

这种方法能够检测来自所有形式细胞死亡的泛性ecDNA。相同的方法和抗体用于人类肾脏活检组织（从第4步）。所有动物和人类科目都获得莫纳什大学和莫纳什健康大学（维多利亚州克莱顿）的伦理批准。
1. 染色的ecDNA与DAPI和β-Actin
<ol><li>在8-10周大的C57/Bl6小鼠中诱导一个20天的抗骨髓性球菌病（抗MPO-GN）模型，对照9。<ol>
<li>使用 CO2腔对小鼠进行人?...

<ol>
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L., Cookson, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-1-dependent+pore+formation+during+pyroptosis+leads+to+osmotic+lysis+of+infected+host+macrophages.">Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages.</a> Cellular Microbiology. 8 (11), 1812-1825 (2006).</li><li>Ooi, J. D., Gan, P. Y., Odobasic, D., Holdsworth, S. R., Kitching, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+mediated+autoimmune+glomerular+disease+in+mice.">T cell mediated autoimmune glomerular disease in mice.</a> Current Protocols in Immunology. 107, 15.27.11-15.27.19 (2014).</li><li>Ruth, 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=Anti-neutrophil+cytoplasmic+antibodies+and+effector+CD4++cells+play+nonredundant+roles+in+anti-myeloperoxidase+crescentic+glomerulonephritis.">Anti-neutrophil cytoplasmic antibodies and effector CD4+ cells play nonredundant roles in anti-myeloperoxidase crescentic glomerulonephritis.</a> Journal of the American Society of Nephrology. 17 (7), 1940-1949 (2006).</li><li>Nagler, M., Insam, H., Pietramellara, G., Ascher-Jenull, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+DNA+in+natural+environments:+features,+relevance+and+applications.">Extracellular DNA in natural environments: features, relevance and applications.</a> Applied Microbiology and Biotechnology. 102 (15), 6343-6356 (2018).</li><li>Burnham, 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=Urinary+cell-free+DNA+is+a+versatile+analyte+for+monitoring+infections+of+the+urinary+tract.">Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract.</a> Nature Communications. 9 (1), 2412 (2018).</li><li>Gobe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+apoptosis+in+kidney+tissue+sections.">Identification of apoptosis in kidney tissue sections.</a> Methods in Molecular Biology. 466, 175-192 (2009).</li><li>Olander, M., Handin, N., Artursson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-Based+Quantification+of+Cell+Debris+as+a+Measure+of+Apoptosis.">Image-Based Quantification of Cell Debris as a Measure of Apoptosis.</a> Analytical Chemistry. 91 (9), 5548-5552 (2019).</li><li>Arganda-Carreras, 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=Trainable+Weka+Segmentation:+a+machine+learning+tool+for+microscopy+pixel+classification.">Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification.</a> Bioinformatics. 33 (15), 2424-2426 (2017).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Apel, F., Zychlinsky, A., Kenny, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neutrophil+extracellular+traps+in+rheumatic+diseases.">The role of neutrophil extracellular traps in rheumatic diseases.</a> Nature Reviews Rheumatology. 14 (8), 467-475 (2018).</li><li>Chapman, E. 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=Caught+in+a+Trap?+Proteomic+Analysis+of+Neutrophil+Extracellular+Traps+in+Rheumatoid+Arthritis+and+Systemic+Lupus+Erythematosus.">Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus.</a> Frontiers in Immunology. 10, 423 (2019).</li><li>Oliveira, V. 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=Sudan+Black+B+treatment+reduces+autofluorescence+and+improves+resolution+of+in+situ+hybridization+specific+fluorescent+signals+of+brain+sections.">Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections.</a> Histology &amp; Histopathology. 25 (8), 1017-1024 (2010).</li><li>Kessenbrock, 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=Netting+neutrophils+in+autoimmune+small-vessel+vasculitis.">Netting neutrophils in autoimmune small-vessel vasculitis.</a> Nature Medicine. 15 (6), 623-625 (2009).</li><li>Schreiber, 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=Necroptosis+controls+NET+generation+and+mediates+complement+activation,+endothelial+damage,+and+autoimmune+vasculitis.">Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (45), E9618-E9625 (2017).</li><li>Antonelou, M., Perea Ortega, L., Harvey, J., Salama, A. 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存在多种方案，用于测量肾上腺炎患者和小鼠模型血清和尿液中的消炎标记物。这种所述协议允许直接分析球状体内的细胞死亡产物（ecDNA、NET和ecMPO）。该协议中最重要的步骤是组织准备和成像。使用荧光染色方法进行分析的主要限制因素是组织自荧光。形式内固定石蜡组织受到自荧光，可以掩盖特定的荧光染色。染色方法的最后一步，其中幻灯片浸入苏丹黑色，衰减组织的自荧光，并允许通过...

骨髓环氧酶抗中性粒细胞质抗体相关血管炎（MPO-AAV）是一种自身免疫性疾病，导致肾功能衰竭的病理球状损伤与相当大的细胞死亡和脱氧核糖核酸（DNA）释放11，2。2DNA可以作为危险信号激活免疫系统。在正常健康条件下，DNA的核位置可以保护他们免受免疫系统的照射。自我DNA在致病过程或自身免疫过程中在细胞外释放，被免疫系统视为一种有效的亲炎损伤相关分子自蛋白（DAMP）3。额外的细胞DNA（ecDNA）通过几个不同的机制从垂死细胞中释放出来，这些机制受不同的生化途径控制，如凋亡、神经球菌外细胞陷阱形成（NET）、坏死或肺尘埃增多4,4、5、6、7、8。5,6,7,8
我们在此描述从死亡细胞释放的ecDNA的方法，从实验抗MPO GN和从MPO-AAV99，1010患者患者体内固定石蜡嵌入（FFPE）肾脏部分释放。从血清和尿液以及体外测定11，12,12检测中检测循环双链DNA（dsDNA）和DNA复合物的方法有多种。这些方法虽然准确确定ecDNA的含量，但不能确定在解剖学上释放ecDNA的位置。有一些方法描述了ecDNA的具体测量，如凋亡调谐器和细胞碎片的测量13，14。13,没有任何方法描述测量ecDNA在发生病理损伤的FFPE肾脏中所有形式的细胞死亡。这一点很重要，以确定实验性治疗是否正在清除从实际目标器官的病理损伤部位的ecDNA。
从肾脏样本中采集多个图像可创建大量数据，通常由一个用户进行分析。这耗费人力，耗时，由于用户偏见，其他用户可能会难以重现。可训练Weka分段是ImageJ的开源软件插件，它使用尖端的生物信息工具使用机器学习算法15、16,16对像素进行分类。此方法是"可训练"的，它根据用户对像素段的分类来学习，并将新学习的分类应用于其他图像。此方法依赖于 ImageJ 程序中的常见分析工具，这些工具用于将段中的每个像素"分类"为属于特定"类"。程序学习"分类器"后，可用于识别同一图像中的其他类似分类段。然后，此模型将保存并应用于同一实验中的其他图像集。
目前确定肾分部原位ecDNA的障碍是内源性自荧光，从正式固定在形式和劳动密集型分析的图像。我们在这里描述如何淬火这种自动荧光，检测ecDNA，并使用监督机器学习进行高通量测量ecDNA。我们之前已经发布了使用 ImageJ 中的宏进行的 NET 和细胞外 MPO （ecMPO） 的测量，现在我们演示了这些方法使用监督机器学习1的半自动化。我们演示了机器学习工具的适应性，以在同一图像中对 NET 和 ecMPO 的替代污渍进行分类。此处描述的这些染色方法用于检测 ecDNA、NET 和 ecMPO，可转换为其他固体器官和疾病，其中 ecDNA、NETS 和 ecMPO 在使类风湿性关节炎和狼疮等,疾病长期存在中发挥作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A2153</td>
			<td>5% and 1% solutions are made up in PBS, can be made in bulk and frozen- discard once thawed.</td>
		</tr>
		<tr>
			<td>Chicken anti Goat IgG (H+L) cross absorbed antibody Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21468</td>
			<td>Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail</td>
		</tr>
		<tr>
			<td>Chicken anti mouse IgG (H+L) cross absorbed antibody, Alex Fluor 647</td>
			<td>ThermoFisher Scientific</td>
			<td>A-121468</td>
			<td>Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail</td>
		</tr>
		<tr>
			<td>Chicken anti rabbit IgG (H+L) Cross absorbed antibody Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21441</td>
			<td>Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail</td>
		</tr>
		<tr>
			<td>Chicken sera</td>
			<td>SIGMA</td>
			<td>C5405</td>
			<td>Made up in 1%BSA/PBS</td>
		</tr>
		<tr>
			<td>Coverslips 24 x60 mm</td>
			<td>Azerscientific</td>
			<td>ES0107222</td>
			<td>#1.5 This is not standard thickness- designed for use in confocal microscopy</td>
		</tr>
		<tr>
			<td>EDTA 10mM</td>
			<td>SIGMA</td>
			<td>E6758</td>
			<td>Add TRIS and EDTA together in distilled water and pH to 9, for antigen retrieval, can be made up in a 10x Solution</td>
		</tr>
		<tr>
			<td>Ethanol 30%, 70% and 100%</td>
			<td>Chem Supply</td>
			<td>UN1170</td>
			<td>Supplied as 100% undenatured ethanol- dilute to 30% and 70% using distilled water</td>
		</tr>
		<tr>
			<td>Formaldehyde, 4% (10% Neutral buffered Formalin)</td>
			<td>TRAJAN</td>
			<td>NBF-500</td>
			<td>Kidney is put into a 5ml tube containing 3ml of formalin for 16 hours at RT, formalin should be used in a fume hood</td>
		</tr>
		<tr>
			<td>Glass histology slides- Ultra Super Frost, Menzel Glaze, 25x75 x1.0mm</td>
			<td>TRAJAN</td>
			<td>J3800AM4</td>
			<td>Using positive charged coated slides is essential. We do not recommend using poly-L-lysine for coating slides as tissue dislodges from slides during the antigen retrieval step</td>
		</tr>
		<tr>
			<td>Goat anti human/mouse MPO antibody</td>
			<td>R&#38;D</td>
			<td>AF3667</td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Histosol</td>
			<td>Clini Pure</td>
			<td>CPL HISTOSOL 08</td>
			<td>Used neat, in 200ml staining rack containers, use in a fume hood</td>
		</tr>
		<tr>
			<td>Hydrophobic pen</td>
			<td>VECTOR Labs</td>
			<td>H-400</td>
			<td>Use to draw circle around kidney tissue</td>
		</tr>
		<tr>
			<td>Mouse anti human/mouse Peptidyl arginase 4 (PAD4)</td>
			<td>ABCAM</td>
			<td>ab128086</td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Nikon C1 confocal scanning laser head attached to Nikon Ti-E inverted Microscope</td>
			<td>Coherent Scientific</td>
			<td></td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>SIGMA</td>
			<td>P38135</td>
			<td>0.01M PB/0.09% NaCl Make up 5L at a time</td>
		</tr>
		<tr>
			<td>Pressure Cooker 6L Tefal secure 5 Neo stainless</td>
			<td>Tefal</td>
			<td>GSA-P2530738</td>
			<td>Purchased at local homeware store</td>
		</tr>
		<tr>
			<td>Prolong Gold DAPI</td>
			<td>Life Technologies</td>
			<td>P36962</td>
			<td>Apply drops directly to coverslip</td>
		</tr>
		<tr>
			<td>Rabbit anti human/mouse Beta Actin antibody</td>
			<td>ABCAM</td>
			<td>ab8227</td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Rabbit anti human/mouse H3Cit antibody</td>
			<td>ABCAM</td>
			<td>ab5103</td>
			<td>Aliquot and freeze at minus 80 degrees upon arrival</td>
		</tr>
		<tr>
			<td>Staining rack 24 slides</td>
			<td>ProScitech</td>
			<td>H4465</td>
			<td>Staining rack chosen has to be able to withstand boiling under pressure and incubation in 60 degree oven</td>
		</tr>
		<tr>
			<td>Sudan Black B</td>
			<td>SIGMA</td>
			<td>199664</td>
			<td>0.3% Add 3g to a 1L bottle in 70% Ethanol, filter and protect from the light- stable for 6 months at room temperature</td>
		</tr>
		<tr>
			<td>Tris 10mM</td>
			<td>SIGMA</td>
			<td>T4661</td>
			<td>Add TRIS and EDTA together in distilled water and pH to 9, for antigen retrieval, can be made up in a 10x solution</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Trajan</td>
			<td>XL005/20</td>
			<td>Must be use used in a fume hood</td>
		</tr>
	</tbody>
</table>

supervised machine learning for semi-quantification of extracellular dna in glomerulonephritis

球状细胞死亡是骨髓过氧化物酶抗中性粒细胞质抗体相关血管炎（MPO-AAV）的病理特征。细胞外脱氧核糖核酸（ecDNA）在不同形式的细胞死亡期间释放，包括凋亡、坏死、坏死、中性粒细胞外陷阱（NETs）和肺气肿。测量这种细胞死亡是耗时与几个不同的生物标志物需要识别不同的生化形式的细胞死亡。ecDNA 的测量通常以血清和尿液作为肾损伤的代理进行，而不是发生在发生病理损伤的实际目标器官中。目前调查肾内肾肾脱氧核糖核酸的难点在于缺乏在实验和存档的人类肾脏活检中采用正式固定石蜡嵌组织（FFPE）的方法。该协议概述了在 FFPE 组织（人类和鼠）中染色 ecDNA 所需的步骤，使用公开提供的开源 ImageJ 插件可训练的 Weka 分段，在生成的图像中测量 ecDNA。可训练的Weka分段应用于球蛋白中的ecDNA，程序学习对ecDNA进行分类。此分类器应用于后续获得的肾脏图像，减少了对每个单个图像进行手动注释的需要。可训练韦卡分段的适应性在肾组织中进一步证明，从实验鼠抗MPO球状体炎（GN），以识别NET和ecMPO，抗MPO GN的常见病理贡献者。该方法对肾组织中的ecDNA进行了客观分析，明确证明了可训练的Weka分段程序能够区分正常肾组织和患病肾组织之间的疗效。该协议可以很容易地适应，以识别在其他器官的ecDNA，NET和ecMPO。

这些图像表示成功使用可训练 Weka 分段所需的多个步骤，以尽量减少从具有诱导抗 MPO GN 的小鼠荧光染色的 FFPE 肾脏组织中对 ecDNA 的劳动密集型人工测量。这些步骤在图 1和图2中总结，其中图像直接取自 Weka 分段程序，概述了分析过程中的每一个步骤。然后，图 3显示了该分析的测量结果，演示了程序在?...

Watch this Scientific Journal Video about Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis at JoVE.com

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

细胞外DNA（ecDNA）在细胞死亡期间释放是消炎，并会导致炎症。在损伤部位测量ecDNA可以确定靶器官治疗的有效性。该协议描述了使用机器学习工具自动测量肾脏组织中 ecDNA。

在球蛋白细胞炎中进行半细胞外DNA半定量的受监督机器学习

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...

肾上腺炎导致病理性肾上腺损伤，细胞大量死亡，细胞外DNA释放。该协议允许在病理损伤期间释放地点的细胞外DNA可视化。这项技术的优点是它测量肾脏细胞死亡的细胞外DNA。与血清或尿液相反，细胞外DNA测量是病理损伤的代理标记。这种方法的意义是，细胞外DNA染色和图像分析都很容易转移到其他自身免疫性疾病，如风湿性关节炎和狼疮。这种方法需要肾脏组织学的基本知识。为了能够准确检测球状细胞外DNA，研究人员必须能够正确识别球状细胞外DNA。对于细胞外DNA染色，首先在60摄氏度的滑架中加热感兴趣的样品60分钟，然后将幻灯片浸入两个40分钟的烟气罩中溶剂变化中。第二次浸入后，在100%乙醇中对样品进行两次补水，每次处理5分钟，在70%乙醇中再补充一次。最后一次治疗后，使用钳子将幻灯片放入高压力锅中放入沸腾的抗原检索溶液中 10 分钟。揭开抗原表名后，将压力锅从热转移到水槽中，并立即将冷自来水放在盖子上。在抗原检索溶液中，让幻灯片平衡 20 分钟，然后每次洗涤时用 0.01 摩尔 PBS 将样品用 0.01 摩尔 PBS 洗涤两次。第二次洗涤后，使用疏水笔在肾脏组织样本周围画圆，并在室温下用 5%牛血清白蛋白中 60 微升的 10% 鸡血清阻止任何非特异性染色，30 分钟。在孵育结束时，在4摄氏度的湿度室中，小心地用60微升的原抗体的阻尼溶液更换。第二天早上，在PBS的轨道摇床上清洗幻灯片两分钟，然后在每张幻灯片中加入60微升适当的荧光素结合二次抗体，在室温下孵育40分钟。在孵育结束时，如演示，在PBS中清洗幻灯片，随后在0.3%苏丹黑色70%乙醇中进行治疗，以淬火任何潜在的形式素引起的自荧光。30 分钟后，用自来水清洗滑梯，清除任何沉淀物，将滑梯浸入 PBS 中 10 分钟，以防止任何进一步的苏丹黑沉淀物形成。在孵化结束时，将滑梯安装到共和玻璃盖玻片上，用三滴 60 微升的安装溶液加用 DAPI，用指甲油密封盖玻片。让幻灯片在室温下固化 24 小时。将它们存放在摄氏四度，防止光线照射，然后根据标准协议通过共声激光扫描显微镜进行成像。请务必用指甲油密封盖玻片，以防止成像过程中的任何移动，并清洁乙醇的盖玻片，以提高成像分辨率。每张幻灯片成像 20 glomeruli 后，打开 ImageJ 中的可训练 Weka 分段插件，然后将第一个图像放到 ImageJ 工具栏上。单击图像、颜色和拆分通道。将显示显示原抗体染色的图像。若要创建单个图像的合并文件，请单击颜色和合并通道。将出现一个弹出窗口，要求将颜色分配给每个通道。选中创建合成并保留源图像框，然后单击"确定"。将显示合并的复合图像。以原抗体染色为指导，在球状塔夫特周围绘制一个感兴趣的区域。要对单个蓝色 DAPI 图像执行高斯模糊，请单击过程、滤镜和高斯模糊。将模糊设置为一对二。图像会变得有点模糊，但核会显得光滑，背景会变软。接下来，单击插件、分段和可训练的 Weka 分段，并使用免费手动工具跟踪完整的核。添加所有感兴趣的核后，添加类 1 框，使用行工具将背景区域划分到类二框。单击"创建新类"并使用线条工具勾勒出 DAPI 染色的核外 DNA 轮廓。单击列车分类器并获取概率。切换鼠标以获取所有组件的黑白图像，选择对象以白色突出显示。要复制此图像，请单击图像并复制。在图像下，调整和阈值。使用鼠标按钮调整阈值，直到只观察到细胞外DNA。在阈值之后，单击进程、二进制，并制作二进制，然后复制以前生成的感兴趣的光大区域。按"控制移位 E"可选择编辑、选择和还原以还原所选内容。要仅测量球状中细胞外DNA颗粒，请单击"分析粒子"和"确定"。将生成一个结果表，显示包含细胞外 DNA 的粒子数、面积、平均像素和球状百分比。若要将分类器另存为细胞外 DNA 分类器，请单击保存分类器并保存数据。若要将模型重新应用到后续图像，请重复初始图像处理，如选择加载分类器和保存的分类器文件之前所示。日志菜单将弹出并运行模型。模型运行后，单击加载数据并选择细胞外 DNA.arff。然后单击"创建结果"。如果生成的图像无法拾取所有核、背景或细胞外 DNA，请单击重新训练分类器并根据需要添加更多分类器。在这里，显示代表性DNA和β-actin染色的单通道图像。合并这两幅图像后，可以绘制一个感兴趣的区域，以测量小鼠肾球状体内的细胞外DNA，如所证明的可训练韦卡分段。该模型可用于识别来自对照患者的肾脏活检标本中的细胞外DNA，或与与肌羚羊体血管炎患者进行肾脏活检的比较。此外，这个程序可以转化为肾脏组织样本中的其他污渍，例如，为嗜中性粒细胞外陷阱染色，并在与安卡相关的血管炎中沉积细胞外肌精。重要的是，当多个用户分析相同数据时，不会观察到显著差异。分析最关键的步骤是提供足够的背景、核和细胞外DNA示例，其中球蛋白作为 Weka 分段程序的分类器。此方法易于传输。例如，可以通过对凋亡或坏死标记物的特定抗体进行染色，进一步分析不同类型的细胞死亡。我们的技术可用于其他炎症过程，如评估自身免疫肾病中的TLR表达或人体肾脏活检中的细胞外肌细胞酶表达。

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

在口腔生物膜细胞外pH值成比例成像

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...