ISX 221128 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.