Light Microscopic and Flow Cytometric Detection of Apoptosis and Necrosis of Neutrophils in the Mammary Gland of the Virgin Heifer

Flow cytometry analysis of discrimination of apoptotic and necrotic neutrophils and their distribution on dot plots using cells influx induced by intramammary administration of buffered saline was verified by light microscopy.Twenty mammary glands of five nonpregnant heifers were used and samples of mammary gland lavages were collected before (control) and 24, 48, 72, and 96 h after treatment and examined by light microscopy and flow cytometry with Annexin V and propidium iodide staining.Verification of flow cytometry method was provided by morphological features of leukocytes in light microscopy and by changes in differential cell counts during induced influx.Detection of apoptotic and necrotic neutrophils was made using backgating technique.There were no significant differences between flow cytometry and light microscopy methods in the percentages of apoptotic neutrophils.By contrary, differences of high significance between methods in the percentages of necrotic neutrophils was determined.It can be concluded that the identification of apoptotic and necrotic neutrophils by flow cytometry is a suitable method for the detection of apoptotic neutrophils of the bovine mammary gland.However, a single trait cannot be regarded as an unambiguous proof of cell necrosis.Gating of a major population of neutrophils seems unsuitable, contrary to backgating, because the majority of apoptotic and necrotic neutrophils was localised out of a major population of neutrophils. Apoptosis, necrosis, neutrophil, mammary gland, bovine