Macrophage migration assay

WT and NOX4KO HSC were isolated and plated in a 24-well plate from a cell migration assay kit (Cell Biolabs, Inc. San Diego, CA). The cells were transfected with a CCR2 expression plasmid DNA (GeneCopoeia, Rockville, MD) using GeneJuice^® transfection reagent, followed by transduction with adeno-CCL2 (Vector Biosystems Inc. Malvern, PA). Control plasmid and virus were used in parallel. Twenty four hours later, HSC were washed and macrophages from WT mice were seeded on the upper chambers and co-cultured with HSC. After 12 hours of co-culture, migration was assessed following manufacturer’s instruction. In brief, the non-migrating cells from the upper side of chambers were removed and the cells migrated to the other side were labeled with a fluorescent dye. The cells were then lysed and the fluorescence intensity was measured at 480 nm/520 nm.