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.

Resuspend the isolated low-density neutrophils at a 5 times 10 to the sixth cells per milliliter of RPMI 1640 medium supplemented with FBS concentration and seed 1 milliliter of neutrophils per well on a 6-well poly-L-lysine coated plate. After 2 hours at 37 degrees Celsius and 5% carbon dioxide, stain the cells in each well with an appropriate fluorescent membrane-impermeable nuclear and chromosomal dye according to the manufacturer's instructions and immediately observe the morphology of the NETs by fluorescence microscopy.

To assess tumor cell adhesion to the NETs, resuspend gastric tumor cells stained with a contrasting fluorescent dye at a 1 times 10 to the sixth per milliliter of RPMI 1640 supplemented with 0.1% bovine serum albumin concentration and add 1 milliliter of tumor cells to each well of the low-density neutrophil cultures. After 5 minutes at 37 degrees Celsius, remove the supernatant from each well and gently wash the cells two times with 2 milliliters of pre-warmed RPMI 1640 medium supplemented with BSA per wash.

A 5-minute incubation is long enough for detecting NET-specific binding. Add the warm medium to the side of each well slowly, gently swirl the plate, and remove the supernatant with an aspirator.

The NETs and the attached tumor cells can then be observed by fluorescence microscopy.