Neutrophil Transepithelial migration assay

3 × 10⁵ pneumocytes were seeded on the basolateral side of a transwell insert with a 3.0 μm pore size membrane by inverting the transwells (Fig. 1a). The setup was incubated at a 37 °C, 5% CO₂ incubator for 6 h to allow cell attachment. After 6 h, the transwells were flipped upright and medium was added back to both apical and basolateral chambers and the monolayer was allowed to grow for at least 48 h into a tight monolayer, confirmed by transepithelial resistance measurements.

Neutrophil transepithelial migration in a primary human type I-like pneumocyte transwell system. a Schematic diagram showing the setup of transepithelial migration. Pneumocytes (Pϕ), indicated as the blue cells, were seeded at the basolateral side of the transwell membrane with a pore size of 3.0 μm. Naïve neutrophils (Nϕ) were added at the apical chamber, as if they infiltrate from the basolateral size of Pϕ to the alveolar space (basolateral chamber) in vivo. b Percentage of neutrophil migration across mock (grey) and influenza virus infected Pϕ layer at 6 and 24 hpi. * indicates p < 0.05 between the indicated group(s)

To infect the pneumocytes, transwells were inverted with the apical surface of the pneumocytes exposed to 80 μl of influenza virus at a multiplicity of infection (MOI) of 0.01 for 1 h followed by PBS wash for three times. The transwells were then returned to its original orientation in a 24 well ultra-low adhesion plate with small airway growth medium (SAGM, Lonza). At 6 and 24 hpi, 5 × 10⁵ naïve neutrophils were added to the apical chamber, and 90 mins were allowed for the transepithelial migration to take place [23]. After 90 mins, the number of neutrophils that migrated though the epithelial monolayer into the lower chamber was counted and expressed in percentage of transmigration.