Bone Marrow Macrophage Response to Vibrio vulnificus

We isolated bone marrow cells and cultured them with 10% L-929 cell culture medium as described previously [15 (link)]. We plated BMMϕs in 35 mm dishes and cultured them for 12 hours. We then added live or heat inactivated V. vulnificus at the indicated multiplicity of infection (MOI) or V. vulnificus lysates to the cells. We collected supernatants at the indicated times for cytokine quantification. We then subjected the infected cells to lysate preparation and Western blot analysis by following a previous protocol [41 (link)]. Anti-phosho-mTOR, anti-phosho-S6K1, anti-phosho-S6, anti-phosho-Akt, anti-phosho-4EBP1, anti-phosho-IKKα/β, anti-phosho-IκBα, and anti-β actin antibodies all came from Cell Signaling Technology.