Cd16b staining

Neutrophils were isolated from the peripheral blood of healthy women and used immediately as previously described (11 ). Briefly, blood was collected into heparin-coated vacutainers (BD Biosciences, San Jose, CA) and neutrophils were isolated by density centrifugation over Histopaque 1077/1119 layers (Sigma-Aldrich). Hypotonic red blood cell lysis was performed and the isolated neutrophil fraction was >95% pure as determined by flow cytometric analysis of CD16b staining (#302005, Biolegend, San Diego, CA, 1:100 dilution). Neutrophils were treated with no treatment (NT; OptiMEM supplemented with 2% FBS alone), untreated FM-CM (FM); LPS-stimulated FM-CM (FM+LPS); or as a control LPS alone (1ng/ml) for 1 hr before removing this media and any associated cytokines, changing the medium to fresh RPMI media with 2% FBS for 4 hrs at 37°C. We have previously demonstrated that this culture protocol does not negatively affect neutrophil viability (11 ). Thus, cell-free neutrophil culture supernatants were collected under sterile conditions and stored at −80°C.

Neutrophils were isolated from the peripheral blood of healthy non-pregnant women and used immediately as previously described (Tong et al. 2019 ). Briefly, blood was collected into heparin-coated vacutainers (BD Biosciences, San Jose, CA) and neutrophils were isolated by density centrifugation on a Histopaque 1077/1119 gradient (Sigma-Aldrich). Hypotonic red blood cell lysis was performed and the isolated neutrophil fraction was >95% pure as determined by CD16b staining (#302005, Biolegend, San Diego, CA, 1:100 dilution).