Image Based Assessment of NFkB Nuclear Translocation in Murine Cells with ImageStream Cytometry

Nuclear Factor kappa B (NFkB) transcription factor plays a central role in regulating many key processes in mammalian cells, including differentiation, cell growth, inflammation, immunity, and stress responses. The activation of NFkB and its translocation from the cytoplasm to the nucleus has become a focal point for studying cellular function, signal transduction pathways, disease mechanisms and drug discovery efforts. The nuclear translocation of NFkB in human cells has been extensively studied with the ImageStream technology during the last decade. However, similar studies in murine cells has not been done. In this project, NFkB nuclear translocation in a murine cell line is measured using ImageStream cytometry. Images of activated and non-activated cells labeled with anti-NFkB antibody and a nuclear dye are acquired on the ImageStream cytometer. Images analysis is performed using IDEAS image analysis software. A Similarity score is determined for every cell based on a pixel by pixel correlation of the nuclear image to the NFkB image. As a result the mean similarity values in the non-activated and activated cell groups are determined. The increased mean similarity value is correlated with higher level of nuclear translocation. The project result will provide a robust method for measuring the NFkB nuclear translocation in the murine cells using ImageStream technology.