Fluorescent plate reader

Macrophages were plated onto 96-well plates (50,000 cells/well), treated with pertussis toxin (PTX) 1 μg/ml overnight or cholera toxin (CTX) (1 μg/ml) for 2 hours at 37 °C. RvD4 (1 nM) or vehicle was incubated with cells for 15 min at 37 °C, followed by addition of BacLight Green-labeled E. coli (2.5×10⁶ CFU) for 60 min at 37 °C. Plates were gently washed, extracellular fluorescence quenched by trypan blue, and phagocytosis determined by measuring total fluorescence (Ex 493/Em535 nm) using a fluorescent plate reader (Molecular Probes). For real-time imaging, MΦ were plated onto 8-well glass chamber slides (100,000 cells/well) and kept in a Stage Top Incubation system for microscopes equipped with a built-in digital gas mixer and temperature regulator (TOKAI HIT model INUF-K14). RvD4 or 17-oxo-RvD4 was added to the cells (10 nM; 15 min at 37°C) followed by BacLight Green-labeled E. coli (5 × 10⁶ CFU). Images were then acquired every 10 min for 2h (37°C) with Keyence BZ-9000 (BIOREVO) inverted fluorescence phase-contrast microscope (40x objective) equipped with a monochrome/color switching camera using BZ-II Viewer software (Keyence, Itasca, IL, USA). Green fluorescence intensity was quantified using BZ-II Analyzer.

Resident peritoneal MΦ were collected from naïve WT and DRV2-KO mice and plated onto 96-well plates (0.5×10⁵ cells/well). RvD2 (1 pM-10 nM) or vehicle controls was incubated with MΦ for 15 min at 37°C, followed by incubation with FITC-labeled serum-treated zymosan particles at 10:1 ratio (zymosan: MΦ) or Baclight Green-labeled E. coli at 50:1 ratio (E. coli : MΦ) for 60 min at 37 °C. Plates were gently washed, extracellular fluorescence quenched by trypan blue, and phagocytosis determined by measuring total fluorescence (Ex 493/Em535 nm) using a fluorescent plate reader (Molecular Probes). The following inhibitors were added together with RvD2 or vehicle control: PKA inhibitor H89 (3μM; Sigma-Aldrich), pERK1/2 inhibitor (50μM; Tocris) and STAT3 inhibitor (100μM; Tocris)

Experiments were conducted as previously described (9 (link)). Macrophages, 7 days differentiated PBMC with GM-CSF (10 ng/ml), were adhered (24 hours) on a 96-well plate (5 × 10⁴ cells/well) in culture medium deprived of GM-CSF. Macrophages were incubated with vehicle (PBS^+/+) alone or compounds (MaR1, 14-oxo-MaR1 or 22-OH-MaR1) for 15 min at 37°C, followed by fluorescent-labeled E. coli (BacLight, Molecular Probes, Carlsbad, CA) at a 50:1 ratio (E. coli:macrophage) for 60 min. Plates were gently washed, extracellular fluorescence quenched by trypan blue, and phagocytosis determined by measuring total fluorescence (Ex 495 nm/Ex 535 nm) using a fluorescent plate reader (Molecular Probes).