In Vitro Adhesion Assay to Detect the Adherence of Cancer Cells to Neutrophil Extracellular Traps

In the cancer microenvironment, activated neutrophils release neutrophil extracellular traps, or NETs, composed of extracellular DNA  fibers, histones, and granular proteins, which play a crucial role in cancer progression.

To study cancer cell-NET adhesion in vitro, add NET suspension to multi-well plate wells. Incubate for NET monolayer formation.

Post-incubation, remove unadhered NETs.

Add a protein solution to block free binding sites in the wells.

Pipette fluorescently labeled cancer cells over NET monolayers.

Specific surface receptors on cancer cells interact with NETs, facilitating cell adhesion.

Post-incubation, discard media from the negative control well.

Add deoxyribonuclease to degrade NETs, detaching the cells.

Remove solutions from negative control and test wells. Use buffer to remove non-adherent cells.

Fix the adherent cancer cells to NETs with formaldehyde.

Using a fluorescence microscope, visualize the fluorescent cancer cells adhered to NETs.

Test wells show significant cancer cell adhesion to NETs, while deoxyribonuclease-treated NETs exhibit reduced levels of cancer cell adhesion.