Hoechst 33342 trihydrochloride dye

Fresh human blood samples from healthy volunteers, aged 18 years and older, were purchased from Research Blood Components (Allston, MA, USA). All blood specimens from patients were obtained with informed consent according to an institutional review board (IRB) approved protocol at the Massachusetts General Hospital. Peripheral blood was drawn into heparinized-tubes (Vacutainer; Becton Dickinson, Woburn, MA, USA) and human neutrophils were isolated within 2 hours after blood collection using EasySep Human Neutrophil Enrichment Kits by following manufacturer instructions (STEMCELL Technologies, Vancouver, Canada). After isolation, neutrophils were washed using cell culture medium, and the nuclei were stained with Hoechst 33342 trihydrochloride dye (Life Technologies, Woburn, MA, USA). Stained neutrophils were then resuspended in medium at a density of 7.5 × 10⁵ cells per mL. Two hundred microliters of cell suspension were then pipetted into each well containing the array of zymosan particle clusters and sealed with a 12 mm diameter coverslip. The purity of the neutrophils was estimated by flow cytometry (using anti-CD66b fluorescent antibodies and Hoechst nuclear stain). Less than 0.1% of the isolated neutrophils had platelets attached to their surface (quantified using anti-CD61 fluorescent antibodies - Supplementary Fig. 10.).