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.</p

NF-kB p65 and STAT1 translocations in TLT macrophages.

<p>a) Percentage of TLT macrophage cells in basolateral co-culture with H4-1 cells in which translocation of STAT1 and NF-kB occurred after treatment with different bacterial strains and positive controls. b) Acquired images of H4-1 cells with cytoplasmic or translocating probes. * indicates significant differences from control cells (p<0.05).</p

NF-kB p65 and STAT1 translocations in small intestinal epithelial cells.

<p>a) Percentage of H4-1 small intestinal epithelial cells in co-culture with TLT macrophages in which translocation of STAT1 and NF-kB occurred after treatment with different bacterial strains and positive controls. b) Acquired images of H4-1 cells with cytoplasmic or translocating probes. * indicates significant differences from control cells (p<0.05).</p

Graphic outline of the study.

<p>By using imaging multicolor flow cytometry we have monitored the translocation of NF-kB p65 and STAT1 in untransformed polarized human neonatal small intestinal epithelial cells H4-1, challenged with different <i>Lactobacillus spp.</i> strains, and in macrophage TLT cells that have been simultaneously co-cultured in a reductionist human 3D model of the immature gut.</p