Tryptase-Mediated Mast Cell Migration Assay

To further investigate effect of tryptase on mast cell migration, a coculture system with HMC-1 and HMEC-1 cells was established. Migration of HMC-1 cells was assessed by using E-16-well plates and the xCELLigence technology (Acea Bioscience, San Diego, CA, USA) [17 (link)]. Briefly, 165 μL medium solutions containing tryptase at 0, 0.3, and 1.0 μg/mL with or without leupeptin (1.0 μg/mL) were added in lower chamber of E-16-well plates, respectively, and incubated for 1 h. For the upper chamber, 30 μL HMEC-1 cells (6 × 10⁴ cells/well) were seeded in, and cells grew for 1 h. Anti-human ICAM-1 antibody (50 μL) was then added in specified wells and cultured for 15 min. This was followed by seeding 50 μL HMC-1 cell suspension (6 × 10⁴ cells/well). All control wells received only the equal volume of medium. The HMC-1 migration number, expressed as a cell index value, was monitored for 48 h. Cells in the lower chamber were collected for flow cytometric analysis of HMC-1. The experiments were conducted in duplicate and repeated 4 times.