Migration of T3M4 cancer cells in a wound-healing assay

kidney medulla The pathophysiological relevance of P2Y14 expression in ICs was assessed by measuring the infiltration of immune cells into the kidney using flow cytometry in mice that were challenged with an injection of UDP-glucose. To address the spatial distribution of immune cells in the kidney, we separated the kidney into cortex and medulla. B1-EGFP mice were challenged with a tail-vein injection of either 100 M MedChemExpress GFT-505 UDP-glucose or saline, as discussed above. Mice were perfused 48 h later through the cardiac left ventricle with PBS to flush the blood out of the kidney vessels and hence measure only tissue infiltrated immune cells. This approach identified a significant and selective accumulation of neutrophils in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19754931 the kidney medulla. This observation is consistent with the upregulation of neutrophil chemo-attractants observed in EGFP cells after UDP-glucose activation. Anti-inflammatory monocytes also slightly decreased in the medulla, but no other significant changes in other immune cell counts were observed. In the kidney cortex, the numbers of most cell types remained unchanged upon UDP-glucose treatment, except for the Ly6C low monocyte population, which decreased slightly. No apparent changes in the number of infiltrated immune 15 / 24 Immune Role of P2Y14 in Intercalated Cells Fig 8. Quantitative PCR detection of pro-inflammatory mediators in MDCK-C11 cells. Detection of mRNA transcripts specific for IL-8, CCL2, CCL4, CCL5, IL1b and TNFa under control conditions and 4h after 100 M UDP-glucose treatment. Data are represented as % changes relative to control. All values are normalized to GAPDH and are shown as Means SEM, P<0.001. Quantification of changes in IL-8 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755349 and CCL2 mRNA expression in MDCK-C11 cells pretreated with the vehicle only or with the MEK inhibitor, PD98059 for 30 minutes in the absence or presence of 100 M UDP-glucose. Quantification of changes in IL-8 and CCL2 mRNA expression in MDCK-C11 cells pretreated with the vehicle only or with PPTN for 30 minutes, in the absence or presence of 100 M UDP-glucose. Data are represented as % changes relative to control. Values are means SEM, P<0.05, P<0.001. doi:10.1371/journal.pone.0121419.g008 16 / 24 Immune Role of P2Y14 in Intercalated Cells Fig 9. Flow cytometry analysis of immune cell infiltration in the kidneys of mice 48 h after injection with saline or 100 M UDP-glucose. Changes in kidney medulla or cortex infiltrated immune cell counts are represented as % changes relative to control. Values are means of percent of each cell population SEM from 46 animals. P<0.05. doi:10.1371/journal.pone.0121419.g009 cells were observed in the cortex and medulla at earlier time points following UDP-glucose injection. The UDP-glucose induced neutrophil infiltration occurs primarily in the renal medulla Kidney sections from control and UDP-glucose treated mice were stained for P2Y14 and for Ly6G, a neutrophil marker. Mosaic images were captured and neutrophils identified based on their positive labeling for Ly6G and their poly-nucleated phenotype . More neutrophils were visible in the medulla of UDP-glucose treated mice compared to sham-treated control mice. Panels A1-A5 and B1-B5 show the individual neutrophils delineated in the circles shown in Panels A and B, respectively. At higher magnification, neutrophils were sometimes seen in the proximity of intercalated cells in the renal medulla of UDP-glucose treated mice, while in control mice, collecting ducts were often seen with no surr