中性粒细胞胞外陷阱
溶菌酶
生物
染色
髓过氧化物酶
达皮
先天免疫系统
白色念珠菌
流式细胞术
亚历山福禄
细胞生物学
化学
生物化学
微生物学
分子生物学
炎症
免疫学
受体
物理
荧光
量子力学
遗传学
作者
Alexia Almaraz-Arreortua,Sorely Adelina Sosa-Luis,William de Jesús Ríos-Ríos,María de los Ángeles Romero-Tlalolini,Sergio Roberto Aguilar-Ruiz,Rafael Baltiérrez-Hoyos,Honorio Torres Aguilar
摘要
Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition. The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (≥ 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.
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