In vitro entry of neutrophils into human bladder epithelial cells. Isolated fresh bovine blood neutrophils were placed on a monolayer of human epithelial cell line 5637 cells (black arrows in A–D) grown on glass cover slides in 24 wells culture plate. After 12 h cells were fixed with PFA and stained with Diff Quick and examined by bright light microscopy. Intraepithelial neutrophils (red arrows in A–C) are visible in bladder cells (black arrows in A–C). Intraepithelial neutrophils are visible in a clear cytoplasmic vacuole displacing the epithelial nuclei. Some epithelial cells contain large numbers of intraepithelial neutrophils (C) while most contain one or two intraepithelial neutrophils Intracellular neutrophils (red arrow in B). PFA-fixed neutrophils fail to internalize and are visible as aggregates of round cells adhered to the epithelial cells (red arrows in D). Scale bars 50 µm (A, D) and 20 µm (B, C). All images are representative of > 3 similar experiments.