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。 2015年12月,90(12):1155 - 8。
DOI：10.1002 / AJH.24185。 EPUB 2015年10月6日。

流式细胞仪测定用于直接定量血液样品中的嗜中性粒细胞细胞外捕集器

玛蒂尔德Gavillet.1, 金伯利马提尼多23, Raffaele Renella134, Chad Harris1, Nate I Shapiro5, Denisa D Wagner123, David A Williams134
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流式细胞仪测定用于直接定量血液样品中的嗜中性粒细胞细胞外捕集器

玛蒂尔德Gavillet.et al. am j hematol.。 20.15.Dec。
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Abstract

中性粒细胞细胞外陷阱（网）有助于先天免疫以及众多疾病过程，如深静脉血栓形成，心肌缺血和自身免疫疾病。迄今为止，通过体外中嗜中性粒细胞的定性显微镜检查，或体内聚集结构的定性微观检查，已经收集了对网的大多数知识。在这里，我们描述了一种新的流式细胞术（流动）基础的测定，以使用针对关键网成分的抗体，特别是DNA，修饰的组蛋白和颗粒酶来鉴定和定量网。该方法适用于鼠和人类样品，用于评估体外诱导的网，或在血液样本中检测体内的净化。通过使用先前证明的两种遗传小鼠模型来验证这种基于流动的方法通过使用先前证明的缺陷的未切割的缺陷。然后在健康的人中性粒细胞上使用，用于检测来自败血症患者的前体内诱导的网和血液样本，用于直接评估体内净胞质中性粒细胞。这种新方法允许对每个样品的数千个细胞进行快速和稳健的评估，并且与潜在的观察者 - 偏差无关，微观定量的两个主要局限性。使用这种新技术有助于直接检测血液样品中的体内循环网，并通过荧光激活的细胞分选（FACS）纯化网上嗜中性粒细胞进行进一步分析。

数据

图1
图1。发展ment and validation of flow assay on mouse samples
A.和B.验证基于流动的测定来测量网。中性粒细胞从外周血中分离出来RAC2.-/−mice or WT controls and stimulated with ionomycin (4 μM), PMA (100 nM) or the same volume of RPMI (Unstim) for 4 hours. Cells were then fixed and stained for NETs, by either microscopy-based assay (A); or by a flow-based assay (B) (N= 4 each). T-test: *P<0.05, **P<0.01, ***P<0.001, versus control (WT Unstim) unless otherwise specified. (C) NETs were detected in PB from WT mice stimulated with ionomycin, but not in PB from Pad4−/− mice or from unstimulated WT controls. Representative flow data showing H3Cit-positive and MPO-positive cells defined as NETs.
图2.
图2.人类样品对流动测定的验证和分析
A. Separation of NETs from resting neutrophils. Neutrophils were purified from peripheral blood of control individuals. NETs were induced by ionomycin for 4h and unstimulated PMNs from the same donor were kept in parallel. Both samples were then mixed 1:1 before fixation, staining, and sorting onto glass slides. Photomicrographs demonstrate the correlation between sorted DAPI+, H3Cit+and MPO+NETs population with the characteristic shooting star morphology of extracellular fibrous DNA used to identify them by microscopy (right panel). Sorted intact PMNs defined as DAPI+, H3Cit-, MPO-exhibit typical multi-lobulated shaped nuclei of resting human neutrophils (left panel). These images are representative from one of two independent experiments. B. Detection of NETs formed in human samples离体要么体内。如上所述加工人EDTA-抗凝整血。一世。从健康供体中的PB诱导网，与未刺激的样品相比。表示初始FSC / SSC印迹的代表性流量数据和被检测为H3CIT和MPO双阳性电池的网。II。（n = 3），III的定量分析。量化体内PB样品中的循环网来自健康对照（n = 7）和败血症患者（n = 5）。Mann-Whitney测试：** P <0.01。
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