The flow cytometric evaluation of phagocytosis by equine peripheral blood neutrophils and pulmonary alveolar macrophages.

Flow cytometry – a technique used to detect and measure physical and chemical characteristics of cells – was successfully applied to investigate how two types of horse cells interact with bacteria. Specifically, researchers focused on peripheral blood neutrophils and pulmonary alveolar macrophages.

Methodology

• The researchers prepared cell populations from venous blood by using a process known as ammonium chloride lysis and also from bronchoalveolar lavage derived samples. Bronchoalveolar lavage is a medical procedure in which a bronchoscope is passed through the mouth or nose into the lungs to obtain cells for examination.
• They then used flow cytometry to differentiate clusters of cells that corresponded to different leucocyte (white blood cell) groups. This differentiation was made possible by the differing light scattering properties of the cells, thus eliminating the need for prior cell separation.
• To identify the bacteria interaction with cells, fluorescent-labelled bacteria were introduced. Cells that could associate with these bacteria, either by attaching them to the cell membrane or by internalizing them, showed enhanced fluorescence.
• To further differentiate the cells based on their interaction with bacteria, substances such as trypan blue and ethidium bromide were added to the sample. If a cell had internalized bacteria, its fluorescence would be either quenched by the trypan blue or counterstained by the ethidium bromide.

Significance

• This method offers a refined way to study bacterial interaction with specific types of cells. The use of flow cytometry enables researchers to single out specific cells and observe how they engage with bacteria.
• The addition of trypan blue and ethidium bromide serves to further distinguish cells, allowing for the identification of cells which internalized bacteria. This highlights how cells react differently to the presence of bacteria, with some simply attaching the bacteria to their surface while others consuming them.
• This research could have implications in studying diseases caused by bacteria, as understanding how cells interact with bacteria at a microscopic level could lead to more targeted and effective treatment strategies.