Neutrophil Migration Assay with Conditioned Media

Neutrophil isolation was performed as previously described (Albrengues et al., 2018 (link)). Transwell migration assays were performed with conditioned media derived from cell lines ectopically expressing p63 or the empty vector that were cultured alone or in combination with mouse PSCs for five days in Matrigel. 5 × 10³ SUIT2 cells and 4 × 10⁴ PSCs were used for the co-cultures. The conditioned media were added to the lower chamber, and primary mouse neutrophils (2.5 × 10⁵) were seeded in 0.5% FBS containing DMEM in the upper chamber of a 3 µm FluoroBlok cell culture insert (08-772-141; Corning) in a 24 well plate. 500 ng of recombinant mouse (250-11; Peprotech) or human (300-11; Peprotech) CXCL1 was added to DMEM with 5% FBS on the day of seeding neutrophils as controls. For experiments with blocking antibodies, 10 μg/ml human CXCL1 (MAB275-100; R and D Systems) or IgG2B isotype control (MAB004; R and D Systems) was added to the neutrophils prior to the addition of the conditioned media. After 24 hr, the FluoroBlok membrane was stained with DAPI (0.05 mg/ml; D1306; Thermo Fisher Scientific) for 5 min, rinsed in water and mounted onto glass slides using mounting media (17985–16; Electron Microscopy Sciences). The number of invading neutrophils was counted in 10 random fields of view using a fluorescence microscope (Leica SP8 Confocal microscope).