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Guinea-pig (intrusive) and mouse (displacement) blastocysts display different cellular mechanisms of implantation. Blastocysts were placed in CMRL-1066 supplemented with either 10 or 20% fetal calf serum, 0.1M L-glutamine and antibiotics and then transferred to dishes previously coated with either Matrigel or type I collagen. After culture for 48 or 72 h, the dishes were processed for transmission electron microscopy. Blastocysts had attached to both extracellular matrices by 48 h. Matrigel elicited minimal trophoblast cell activity. Trophoblast cell projections were oriented parallel to the Matrigel and displayed little invasive activity, but trophoblast cells displayed active interaction with type I collagen. By 72 h, trophoblast cells exhibited slender, anastomosing projections which extended into the collagen matrix. Bundles of microfilaments running parallel with the long axis of the projections were observed. The morphology of type I collagen was altered in the immediate vicinity of the trophoblast projections. The projections interdigitated and desmosomes developed between processes. Projections appeared to meet, fuse and entrap matrix. These results suggest that trophoblast cells do not significantly interact with Matrigel, but penetrate into type I collagen.

Polymorphonuclear neutrophils (PMNs) have multiple functions during the resolution of inflammation. At the inflammation site, increases in chemokines induce infiltration of PMNs via CXCR receptor activation, resulting in cell clearance via phagocytosis and NETosis. Here we describe characterization of the activation and function of PMNs using IncuCyte® live-cell analysis. Changes in cell shape and CD marker expression are known indicators of PMN activation. Freshly isolated PMNs were seeded in 96-well plates, in the presence of FabFlour-488 labeled CD11b antibody for live-cell immunocytochemistry. Phase and fluorescence images were captured every 30 min for 6 h with IncuCyte S3. Cell-by-cell analysis enables individual cell segmentation and yields area, shape (eccentricity) and fluorescent intensity metrics. Subsets of PMNs could be analyzed by classifying on each of these parameters. CXCL8 induced a time- and concentration-dependent increase in eccentricity (EC50 0.63 nM) and concomitant increase in CD11b expression (EC50 0.22 nM). CXCL8-induced activation was attenuated by an anti-CXCL8 antibody. CXCL1 or CCL2 yielded little or no change in either parameter. As a follow up, we assessed CXCL8-dependent chemotaxis using IncuCyte ClearView Chemotaxis plates, phagocytotic activity with IncuCyte pHrodo® E. coli Bioparticles® and PMA-induced NETosis (IncuCyte Cytotox Green). In all three cases robust, time-dependent signal changes were observed, consistent with known PMN function. We conclude that live-cell analysis is a flexible and powerful method for analyzing neutrophil activity, where morphological, protein and functional parameters can be readily quantified and integrated over time.