Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Observe Adhesive Interaction Between Cancer Cells and NETs

Under pathological conditions like cancer, activated neutrophils release neutrophil extracellular traps or NETs – a mesh of extracellular DNA with associated enzymes and antimicrobial proteins – that entrap circulating cancer cells.

To visualize the adhesive interaction between NETs and tumor cells in vitro, begin by culturing low-density neutrophils, a neutrophil subtype, in a polymer-coated culture dish that promotes cell attachment. Incubate for a few hours to stimulate the neutrophils to form web-like NETs.

Add a suspension of red fluorescein-labeled cancer cells to the neutrophil culture. Incubate the cancer cells with NETs for a brief period to facilitate cancer cell-NET interaction. The NET-DNA acts as a chemotactic factor that attracts cancer cells and promotes their adhesion to NETs.

Remove the spent medium. Gently wash the culture with a suitable fresh medium to remove any unattached tumor cells from the culture well.

Now, add a membrane-impermeable green fluorescent nuclear stain to color the extracellular NETs selectively. Observe the culture under a fluorescence microscope. Neutrophil extracellular traps appear as green string-like structures that form a mesh to which red tumor cells are attached.