Poly l lysine coated glass coverslips

Human placental macrophages (PMs) and fetal membrane tissues were isolated from placental tissue samples from women who delivered healthy infants at full term by cesarean section (without labor). Deidentified tissue samples were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute. All tissues were collected in accordance with the guidelines of the Vanderbilt University Institutional Review Board (approval 131607). Macrophage isolation occurred as previously described (89 (link)); briefly, placental villous tissue samples were minced followed by digestion with DNase, collagenase, and hyaluronidase (all from Sigma-Aldrich, St. Louis, MO). Cells were filtered and centrifuged, and CD14⁺ cells were isolated using the magnetic MACS Cell Separation system with CD14 microbeads (Miltenyi Biotec, Auburn, CA). Cells were incubated in RPMI 1640 medium (ThermoFisher, Waltham, MA) with 10% charcoal stripped fetal bovine serum (ThermoFisher) and 1% antibiotic/antimycotic solution (ThermoFisher) overnight at 37°C in 5% carbon dioxide. The following day, PMs were suspended in RPMI 1640 medium without antibiotic/antimycotic and distributed into polystyrene plates. Cells were incubated for at least 1 h prior to infection to allow for cell adherence to the plate or to poly-L-lysine-coated glass coverslips (Corning, Bedford, MA) for microscopy assays.

Neutrophils (10⁵/well) were seeded in culture dishes with poly-L-lysine-coated glass coverslips (Corning, USA), and incubated with 100 nM PMA or Leishmania promastigotes (1 parasite/1 neutrophil ratio) for 90 min at 35 ^oC with 5% CO₂ and fixed in 4% formaldehyde. Following, slides were stained with primary antibodies anti-Argonaute-2 and -Apoliprotein-AI (Abcam, USA), anti-elastase (Calbiochem, USA) or anti-DNA/histone H1 (Millipore, USA), followed by secondary antibodies goat-anti-rabbit or anti-mouse Alexa Fluor 488 or 546 (Molecular Probes, USA). The slides were mounted in ProLong Gold Antifade Mountant with DAPI (Thermo Fisher). Confocal images were taken in a Zeiss LSM 710.