Complete entry of neutrophils in murine mammary epithelial cell line EPH4 (A, B) and human bladder epithelial cell line 5637. Isolated fresh bovine blood neutrophils were placed on a monolayer of EPH4 cells or human bladder epithelium cell line 5637 grown on glass cover slides in 24 wells culture plate. To ascertain complete entry of neutrophils into the EPH-4 cells and loss of membrane invagination, the epithelial monolayer with intraepithelial neutrophils (white arrow in A) were assayed for protection from surface biotinylation (white arrow in B). PFA-fixed monolayers were stained with phalloidin (red in A) and DAPI (blue in A, B) for epifluorescence and confocal microscopy. The xy image is on the plan indicated by the horizontal and vertical dashed lines shown in the xz and yz images, respectively. Intraepithelial neutrophils were protected from surface labeling with biotin (white arrow in B), indicating their complete entry while crawling and invaginating neutrophils (yellow arrows in A, B) are labeled. A, B Original magnification ×63. To further examine intraepithelial neutrophils in human bladder epithelium cell line 5637, single cell suspension of trypsinized monolayer with intraepithelial neutrophils was cytospun onto glass slides, fixed with 2% PFA and stained with DAPI and phalloidin (B). Analysis by confocal microscopy demonstrated complete entry of neutrophils within epithelial cells or, in some cases, one neutrophil within an epithelial cell that was within a third epithelial cell (C). Scale bar 4 µm (C). All images are representative of > 3 similar experiments.