Ficoll cushion

Preparation of hMDMs and THP-1 cells was described previously^{57 ,58} . Human peripheral blood was obtained from healthy donors with written informed consent via venipuncture following a protocol approved by Nationwide Children’s Institutional Review Board. Peripheral mononuclear cells (PBMCs) were isolated from heparinized blood using a Ficoll cushion (GE Healthcare Bio-Science, Piscataway, NJ, USA). The PBMCs were then washed with RPMI 1640 plus L-glutamine (Gibco-Life Technologies, Grand Island, NY, USA). PBMCs were cultured in Teflon wells with RPMI 1640 plus L-glutamine containing 20% human AB serum (Sigma Aldrich, P2918) at 37°C for 5 days. hMDMs were recovered from Teflon wells and plated at 2×10⁵ cells/well in a 24-well tissue culture plate in the presence of 10% human AB serum for 2 h at 37°C. Lymphocytes were then washed away leaving a hMDM monolayer. The hMDM monolayer was maintained in RPMI supplemented with 10% human AB serum for 4 days before used in Salmonella infection experiments. hMDMs and THP-1 infection with S. Typhimurium and Western blot analysis were performed as previously described⁵⁹ . Macrophage cell lysates were collected at 4 h post-infection and subjected to Western blot for GNLY (R&D Systems Cat# AF3138-SP; 1:500 dilution), CCL20 (R&D Systems Cat# AF360-SP; 1:500), and/or actin (Abcam Cat# 179467; 1:1000) using 40 μg of total protein.