Time-lapse Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Analyze Post-adhesion Interaction between Cancer Cells and NETs
Transcription
During pathological conditions like cancer, activated neutrophils release neutrophil extracellular traps or NETs – a network of extracellular DNA fibers associated with histones and proteolytic enzymes. These structures entrap cancer cells and play a key role in cancer metastasis.
To study the post-adhesion behavior of entrapped cancer cells in NETs in vitro, first, culture low-density neutrophils – a neutrophil subpopulation – in a polymer-coated culture dish that facilitates cell attachment. Incubate to activate the neutrophils to release mesh-like NETs.
Next, add a membrane-impermeable green fluorescent nuclear dye to stain the DNA fibers in NETs specifically. Remove the spent medium. Add a suspension of cancer cells to the neutrophil culture.
Incubate the culture for a short period to facilitate cancer cell-NET interaction. The DNA component of NETs functions as a chemotactic factor that stimulates the migration of cancer cells and aids in their adhesion to NETs.
Remove the spent medium. Gently rinse the culture with appropriate fresh media to remove any unbound cancer cells from the culture dish. Replenish with a fresh serum-containing media.
Image the culture for an extended duration using time-lapse video microscopy to analyze the interactive events between NETs and cancer cells. Over time, the NETs provide a suitable microenvironment facilitating the proliferation of cancer cells. Use suitable software to observe the proliferation of cancer cells on green fluorescent NETs.
