Leukocyte Transmigration Assay

Inserts from 24-well transwell plates (Costar, Sigma-Aldrich) were coated with collagen type 1 (Advanced Biomatrix) and fibronectin (Merck Millipore) (10 μg/ml each) and seeded with 175'000 imLECs in imLEC medium on the lower side or on the upper side of the transwell membrane, in analogy to the setup originally described by Johnson et al. (52 (link)). The medium was exchanged after 6 h and again the next day. Two days later, 1 × 10⁵ LPS-activated BM-DCs (WT or ALCAM^−/−) were added to the top well and left to migrate toward the bottom well containing 100 ng/ml of CCL21 (PeproTech) in DMEM/F12 medium containing 2% FCS. After 4 h, transmigrated DCs were stained with anti-mouse CD11c-APC (BioLegend) and quantified by FACS. For assessing antibody efficacy, WT DCs and LECs were both pre-incubated with I/F8-Fc or KSF-Fc (10 μg/ml) for 30 min at 37°C prior to performing the experiment in presence of antibodies.