ISX 221010 Run Files

Peripheral human neutrophils were isolated as above and pretreated with 50 μg/mL of purified human THP, sialic acid (500 ng/mL, catalog no. A0812, Sigma Aldrich), or mock-treated, and incubated at 37°C in 5% CO2 for 30 min, and then incubated for an additional 2.5 h with PMA (25 nM). The following antibodies (0.5 μg/mL) and dyes (concentrations provided below) were added: Anti-MPO-FITC (clone M1/70, catalog no. 553310; BD Biosciences), Fc Block, Live/Dead Near IR (1:200 of stock, catalog no. L34975; Thermo Fisher Scientific), Sytox orange (100 nM, catalog no. S34861; Thermo Fisher Scientific), and Hoechst 33342 (200 nM, catalog no. 62249; Thermo Fisher Scientific). After a 30-minute incubation on ice, samples were washed 1X, resuspended in a 1:1 mixture of fresh FACS buffer and PBS. Cell morphology via imaging flow cytometry was assessed as described previously (PMID: 30985081). An Amnis ImageStream X Mark II imaging flow cytometer was used for data acquisition with a 60X objective, low flow rate and high sensitivity, and 405, 488, 561 and 635 nm lasers set to 120, 150, 100 and 150 mW respectively. Data were analyzed using the IDEAS version 6.3 software package. Clipped images were retained due to the large size of NETs, and single stained controls were used for compensation. Masking was performed using the default “object (tight)” and “morphology” algorithms as described (PMID: 30985081). Statistics reports were generated using IDEAS and processed IDEAS data analysis files (.daf) were then analyzed in FCS Express 7 to generate analysis plots.按前述方法分离人外周血中性粒细胞，随后分别以50 μg/mL纯化的人THP、500 ng/mL唾液酸（货号A0812，Sigma Aldrich）进行预处理，或设置模拟处理对照组（mock-treated），于37℃、5% CO₂条件下孵育30分钟，之后加入25 nM佛波酯（PMA）继续孵育2.5小时。随后添加浓度为0.5 μg/mL的下述抗体及指定浓度的染料（浓度详见下文）：抗MPO-FITC（克隆号M1/70，货号553310，BD Biosciences）、Fc阻断剂（Fc Block）、死活细胞近红外染料（Live/Dead Near IR，原液按1:200稀释，货号L34975，Thermo Fisher Scientific）、Sytox Orange染料（100 nM，货号S34861，Thermo Fisher Scientific）以及Hoechst 33342染料（200 nM，货号62249，Thermo Fisher Scientific）。冰上孵育30分钟后，将样品以1×洗涤，并重悬于新鲜FACS缓冲液与磷酸盐缓冲液（PBS）按1:1体积比混合的溶液中。采用成像流式细胞术（imaging flow cytometry）检测细胞形态，具体操作参照此前发表的研究（PMID: 30985081）。数据采集使用Amnis ImageStream X Mark II成像流式细胞仪，配置60倍物镜，采用低流速、高灵敏度模式，405 nm、488 nm、561 nm及635 nm激光器的功率分别设置为120 mW、150 mW、100 mW及150 mW。数据分析采用IDEAS 6.3版本软件包完成。由于中性粒细胞胞外陷阱（NETs，Neutrophil Extracellular Traps）体积较大，故保留截切图像；并采用单染对照进行荧光补偿校正。掩膜处理采用默认的"object (tight)"与"morphology"算法，具体操作参照前述文献（PMID: 30985081）。统计报告通过IDEAS软件生成，随后将经IDEAS处理的数据分析文件（.daf格式）导入FCS Express 7软件进行分析并绘制分析图表。