BACKGROUND Neutrophil extracellular traps (NETs), extracellular structures composed of decondensed chromatin and antimicrobial molecules, are released in a process called NETosis. NETs, which are part of normal host defense, have also been implicated in multiple human diseases. Unfortunately, methods for quantifying NETs have limitations which constrain the study of NETs in disease. Establishing optimal methods for NET quantification holds the potential to further elucidate the role of NETs in normal and pathologic processes. RESULTS To better quantify NETs and NET-like structures, we created DNA Area and NETosis Analysis (DANA), a novel ImageJ/Java based program which provides a simple, semi-automated approach to quantify NET-like structures and DNA area. DANA can analyze many fluorescent microscope images at once and provides data on a per cell, per image, and per sample basis. Using fluorescent microscope images of Sytox-stained human neutrophils, DANA quantified a similar frequency of NET-like structures to the frequency determined by two different individuals counting by eye, and in a fraction of the time. As expected, DANA also detected increased DNA area and frequency of NET-like structures in neutrophils from subjects with rheumatoid arthritis as compared to control subjects. Using images of DAPI-stained murine neutrophils, DANA (installed by an individual with no programming background) gave similar frequencies of NET-like structures as the frequency of NETs determined by two individuals counting by eye. Further, DANA quantified more NETs in stimulated murine neutrophils compared to unstimulated, as expected. CONCLUSIONS DANA provides a means to quantify DNA decondensation and the frequency of NET-like structures using a variety of different fluorescent markers in a rapid, reliable, simple, high-throughput, and cost-effective manner making it optimal to assess NETosis in a variety of conditions.

中文翻译：

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DNA 面积和 NETosis 分析 (DANA)：一种在荧光显微镜图像中量化中性粒细胞胞外陷阱的高通量方法。


背景中性粒细胞胞外陷阱（NET）是由解浓缩染色质和抗菌分子组成的胞外结构，在称为 NETosis 的过程中释放。 NETs 是正常宿主防御的一部分，也与多种人类疾病有关。不幸的是，量化 NET 的方法存在局限性，限制了 NET 在疾病中的研究。建立 NET 量化的最佳方法有可能进一步阐明 NET 在正常和病理过程中的作用。结果 为了更好地量化 NET 和类 NET 结构，我们创建了 DNA 区域和 NETosis 分析 (DANA)，这是一种基于 ImageJ/Java 的新颖程序，它提供了一种简单的半自动化方法来量化类 NET 结构和 DNA 区域。 DANA 可以同时分析许多荧光显微镜图像，并提供每个细胞、每个图像和每个样本的数据。使用 Sytox 染色的人类中性粒细胞的荧光显微镜图像，DANA 量化了类似 NET 结构的频率，该频率与两个不同的个体通过肉眼计数确定的频率相似，而且只用了一小部分时间。正如预期的那样，DANA 还检测到与对照受试者相比，类风湿性关节炎受试者的中性粒细胞中 NET 样结构的 DNA 面积和频率有所增加。使用 DAPI 染色的小鼠中性粒细胞图像，DANA（由没有编程背景的个人安装）给出了类似 NET 结构的频率，与两个人通过肉眼计数确定的 NET 频率相似。此外，正如预期的那样，与未刺激的小鼠相比，DANA 量化了受刺激的小鼠中性粒细胞中更多的 NET。 结论 DANA 提供了一种使用各种不同荧光标记以快速、可靠、简单、高通量和经济有效的方式量化 DNA 解缩和 NET 样结构频率的方法，使其成为评估各种 NETosis 的最佳方法。的条件。