Expression of Mf-markers and NF-κB subunits in co-cultures of HSC-3 cells and M1 or R848 Mfs.

Vybrant CM-Dil labeled HSC-3 cells (red) and unlabeled M1- or R848 Mfs were incubated with DMSO or 10 ng/ml TNF-α for 30 min where after cells were fixed for immunofluorescence with antibodies for CD68, CD163, NF-κB p50 and p65 subunits. Some samples were pre-incubated with 10 μM BAY 11-7082 prior to TNF-α activation. AlexaFluor488-conjugated secondary antibody was used for visualization. Samples were mounted with DAPI- mountain medium to visualize nuclei (blue). Samples were photographed with a Leica Confocal microscope with 63x oil immersion objective. CD163 and NF-κB p65 immunofluorescence from HSC-3 cell and M1 Mf co-cultures (A). CD68 and NF-κB p50 immunofluorescence from HSC-3 cell and R848 Mf co-cultures (B). Red arrows indicate HSC-3 cells and green arrows Mfs. Scale bars 50 μm.