Simultaneous Measurement of NF-kB Translocation and Apoptosis With the ImageStream® Multispectral Cell Imaging System

The ImageStream system combines the capabilities of microscopy and flow cytometry in a single platform for quantitative image-based cellular assays in large and heterogeneous cell populations. Here we demonstrate the simultaneous measurement of druginduced apoptosis and NF-kB nuclear translocation using a combination of intensity-based and morphometric parameters. Apoptotic and necrotic cells are identified on the basis of intracellular caspase-3 staining and nuclear morphology, while the location of NF-kB relative to the nucleus is measured using a novel application of image correlation provided with the analysis software. These results demonstrate the power of analytical morphometry in the quantitation of apoptosis and nuclear translocation. The ImageStream is operationally similar to a flow cytometer but it has the ability to simultaneously capture six images of each cell -at a rate of approximately 300 cells per second -with a resolution similar to that of a fluorescence microscope. Each cell is represented by a brightfield image, a darkfield image and up to four different fluorescence images. Using these data, the ImageStream provides quantitative information not just about the intensity of target molecules, but about their location within the cell as well. In addition, the system delivers statistically significant assessments of the cellular morphology of different populations. The combination of these flow cytometry and microscopy capabilities allows a new level of integrated quantitative analysis, both of the individual cell and the cell population. The ImageStream System