John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
Curr Protoc Mouse Biol. 2020 Sep;10(3):e78. doi: 10.1002/cpmo.78.
One of the most intriguing functions of neutrophils is the production of neutrophil extracellular traps (NETs), which are formed when neutrophils decondense their internal DNA and extrude it along with cytotoxic proteins in a web-like structure. This process allows neutrophils to trap and kill pathogens, and is also associated with multiple hematological and autoimmune conditions. Due to their rapid degradation, there are many challenges in accurately and specifically detecting and quantifying NETs. Microscopy is the gold standard for NET detection, but is not optimal for large-scale screening. Furthermore, methods relying on detection of free DNA or on flow cytometry-based examination of NET-associated markers can be nonspecific, time-consuming, and expensive. Here, we describe an innovative, quick, specific, and inexpensive conventional flow cytometry method for detecting neutrophils on the verge of forming NETs. These methods utilize pulse-shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, a prerequisite for NET formation. An increase in DNA-diffuse neutrophils is found in cell populations after exposure to NET-inducing stimuli, consistent with the DNA decondensation expected during neutrophil NET formation. These populations are only observed in granulocytes, validating the specificity of this method. We describe protocols optimized for neutrophils retrieved from mouse blood, spleen, and bone marrow. The relative speed and simplicity of the method described here makes it a useful tool for detecting NET formation in large-scale experiments. © 2020 Wiley Periodicals LLC. Basic Protocol: Detection of nuclear decondensation in neutrophils from stimulated murine bone marrow Alternate Protocol 1: Detection of nuclear decondensation in neutrophils from splenocytes Alternate Protocol 2: Detection of nuclear decondensation in neutrophils from blood Support Protocol 1: Cryopreservation and defrosting of samples Support Protocol 2: Paraformaldehyde fixation of samples.
中性粒细胞的最有趣功能之一是产生中性粒细胞胞外陷阱 (NETs)，当中性粒细胞解凝聚其内部 DNA 并沿着网状结构挤出细胞毒性蛋白时，就会形成 NETs。这个过程使中性粒细胞能够捕获和杀死病原体，并且与多种血液学和自身免疫条件有关。由于其快速降解，因此在准确和特异性检测和定量 NETs 方面存在许多挑战。显微镜检查是 NET 检测的金标准，但不适合大规模筛选。此外，依赖于游离 DNA 检测或基于流式细胞术的 NET 相关标志物检测的方法可能是非特异性的、耗时的且昂贵的。在这里，我们描述了一种新颖的、快速的、特异性的和廉价的传统流式细胞术方法，用于检测处于形成 NET 边缘的中性粒细胞。这些方法利用脉冲状分析 (PulSA) 来区分静止的中性粒细胞和 DNA 解凝聚的中性粒细胞，这是 NET 形成的先决条件。在暴露于 NET 诱导刺激后，细胞群中的 DNA 弥散中性粒细胞增加，与 NET 形成期间预期的 DNA 解凝聚一致。这些群体仅在粒细胞中观察到，验证了该方法的特异性。我们描述了从鼠血、脾和骨髓中回收的中性粒细胞优化的方案。这里描述的方法相对快速且简单，使其成为在大规模实验中检测 NET 形成的有用工具。© 2020 威立出版社。基本方案：检测刺激鼠骨髓来源的中性粒细胞中的核解凝聚备选方案 1：检测脾细胞来源的中性粒细胞中的核解凝聚备选方案 2：检测血液来源的中性粒细胞中的核解凝聚支持方案 1：样品的冷冻保存和解冻支持方案 2：样品的多聚甲醛固定
