MOESM9 of Intraepithelial neutrophils in mammary, urinary and gall bladder infections

Additional file 9. Viability is essential for entry of neutrophils into mammary epithelial cells. Fresh viable, PFA-fixed and UV-treated apoptotic neutrophils were layered over a monolayer of polar mammary epithelial cell line EPH4 and evaluated microscopically for neutrophil internalization after 24 h of co-culture as described in the materials and methods. Neutrophils were cytospun onto glass slides and stained with Diff Quick for microscopic evaluation (A). Neutrophil viability and apoptosis were quantified using FACS analysis following staining with propidium iodide (PI in B) and Annexin V FITC (C). Mean percentage (± SD) of epithelial cells with internalized cells following co-culture with viable, fixed and UV-treated apoptotic neutrophils is presented in D. Results of a representative experiment out of three. Mean % cell-in-cell following co-culture with viable neutrophils was compared by unpaired t test using GraphPad Prism 6 (GraphPad Software, Inc.) and was significantly different from all other groups; *P < 0.005. Scale bars 10 µm (A).