Clinical Immunology Research Department of Biochemical Sciences Faculty, Universidad Autónoma "Benito Juárez" de Oaxaca.
Department of Molecular Biomedicine, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional.
J Vis Exp. 2022 Nov 4(189). doi: 10.3791/64522.
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.
中性粒细胞通过多种机制发挥作为先天免疫反应中细胞防御的第一道防线的功能，例如形成中性粒细胞胞外陷阱（NETs）。本研究使用标准化的体外方法分析微生物和化学刺激物诱导的 NET 的形态和组成变化，用于人类细胞的 NET 诱导和特征分析。这里描述的程序允许分析 NET 的形态（裂解或非裂解）和组成（DNA-蛋白质结构和酶活性），以及可溶性因子或细胞接触对这些特征的影响。此外，这里描述的技术可以进行修改，以评估外源性可溶性因子或细胞接触对 NET 组成的影响。应用的技术包括使用双密度梯度（1.079-1.098 g/mL）从人外周血中纯化多形核细胞，保证最佳纯度和活力（≥95%），如 Wright 染色、台盼蓝排除和流式细胞术所示，包括 FSC 与 SSC 分析和 7AAD 染色。NET 形成是通过微生物（铜绿假单胞菌、金黄色葡萄球菌和白色念珠菌）和化学（佛波醇肉豆蔻酸酯、HOCl）刺激物诱导的，通过 DNA-DAPI 染色、抗菌肽 cathelicidin（LL37）的免疫染色和酶活性（中性粒细胞弹性蛋白酶、组织蛋白酶 G 和髓过氧化物酶）的定量来表征 NET。通过荧光显微镜采集图像，并使用 ImageJ 进行分析。
