CHARACTERIZATION OF NF-¿B DEFICIENT BONE-MARROW MACROPHAGES

Abstract

The aim of this study was tocharacterize bone-marrow derived macrophages (BMDMs) that lack canonical nuclear factor-kappaB (NF-?B) signaling. The macrophages for the study were obtained by harvesting the bone-marrow fromp65LysMCre (KO) mice and LysMCrecontrolmice.To determine NF-?B deletion efficiency, p65 (a transcription factor in the canonical pathway) protein levels were evaluated by fluorescent microscopyin bothKOand control BMDMs that had been stimulated withlipopolysaccharide(LPS).The induction ofiNOSwasmonitoredin KO and control BMDMswhenactivated by NF-?B stimulatorsIFN-?andLPS.The regulation of iNOSwas assessedby comparing macrophages that had been treated withLPS, IFN-?, or both to a control treatment under fluorescent microscopy. In addition to staining, a nitric oxide assay was employed to help determine the extent of iNOS activity.The macrophages were also visualized under light microcopyby comparing macrophagesthat were stimulated with LPS andIFN-?tounstimulated cellsusing fluorescence microscopy.Currently,a caspase assay is in progress to help further evaluate the effects of p65 losswithin macrophages.