Bone marrow-derived macrophage (BMDM) culture and chemotaxis assay

Briefly, BMDMs from femoral bones of 8-wk-old male mice were cultured in BMDM growth medium [RPMI-1640:L929 supernatant: FBS = 5:3:2 (vol/vol) supplemented with 1% (vol/vol) penicillin and streptomycin] for 1 week, and the cells then were stimulated with indicated cytokines (100 ng/mL LPS (sigma), 2 ng/ml IL-6 (R&D), 10 ng/ml IL-1β (R&D), or 10 ng/ml IL-4 (R&D)) or conditioned medium from IECs for the indicated time.

The chemotaxis assay was performed in 24-well inserts with 8-μm pores (Corning, Inc., Corning, NY, USA). BMDMs or RAW264.7 cells (4 × 10⁴/well in 200 μL blank medium supplemented with 0.2% (g/ml) BSA) were seeded in the upper side of each insert and 600 μL of conditioned medium from IECs was added to the bottom well. Following incubation for 24 h, the migrating cells were fixed with 4% (g/ml) polyoxymethylene (PFA, sigma) for 30 min and stained with 0.1% (g/ml) crystal violet solution for another 20 min. Remaining cells on the upper side of insert were removed. Following drying at room temperature, images were captured using an Olympus IX51 microscope and the numbers of cells were counted using ImageJ software.