Analysis of cell populations with imaging flow cytometry.

<p>Cells were stained with Cy3-labeled NF-kB p65 as well as 7AAD and Alexa Fluor 647-labeled STAT1. Images were acquired using the ImageStreamX multispectral imaging flow cytometer, collecting 5000 events per sample at 40× magnification and analyzed using IDEAS image-analysis software. Gating on bivariate plot of aspect ratio versus cell area was first used to isolate a population of single cells. Cells within the focal plane were further selected using a two-dimensional plot of image contrast versus root-mean-squared (rms) gradient. The software compared the location of probes (NF-kB, STAT1) with the location of the nucleus in each acquired cell, running a probe similarity algorithm.