Calcein

Primary neutrophils and epithelial cells were isolated from the lungs of wild type and Ormdl3 transgenic mice using magnetic bead cell selection for Ly6G-positive or CD326 (EpCAM)-positive cells, respectively exactly as we previously described (18 (link)). Epithelial cells were stimulated for 20 h with vehicle or LPS (200 ng/mL) and the neutrophil attachment assay was performed as previously described (18 (link)). Transwell inserts (pore size: 5.0 μm; Sarstedt) were used to measure the migration of neutrophils and pre-coated with laminin. 1.5 × 10⁵ cells were seeded in the top well in serum-free DMEM and placed on wells containing 100 nM fMLP with S1P (100 nM) or LPS (200 ng/ml) in the lower chamber. The chambers were incubated at 37 °C in 5% CO₂ for 1 h. Subsequently, migrated neutrophil numbers in lower chambers were determined by removing all cells from the upper chamber and filling the bottom chamber with 100 μl of cell dissociation solution containing 2 μg/ml Calcein-AM (Life Technologies, CA). The re-assembled chamber was incubated for an additional 1 h at 37°C. Migrated cells in the fluid of the bottom chambers were quantified by measuring fluorescence of Calcein at 485 nm excitation, 520 nm emission with a TECAN Infinite M1000 fluorescence plate reader (Männedorf, Switzerland).