Time-lapse Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Analyze Post-adhesion Interaction between Cancer Cells and NETs

1. Time Lapse Video Analysis of the Trapped Tumor Cells

1. Culture the peritoneal LDNs in a 35 mm round dish coated with poly-L-lysine and incubate for 2 h at 37 ˚C in 5% CO₂.
2. Add the green-fluorescent dye for staining the nucleus and chromosomes to visualize NETs at the final concentration of 5 µM.
3. After 1 min of incubation, remove the media and add 2 mL of 0.1% BSA + RPMI 1640.
4. Remove the media and add unstained 1 x 10⁶ MKN45 cells suspended in 0.1% BSA + RPMI 1640. Incubate for 5 min at RT.
5. Remove any unattached tumor cells by gently washing the dish by adding 2 mL of prewarmed media (0.1% BSA + RPMI 1640) and swirling the dish.
6. Repeat the above washing procedure twice.
   NOTE: Since NETs weakly attach to the plate, washing should be done as gently as possible to avoid removal of the NETs themselves.
7. Add DMEM with 10% FCS supplemented with 100 u/mL Penicillin and 100 µg/mL streptomycin.
8. Mount the culture dish in a whole-view cell observation system and select the appropriate field in which tumor cells are trapped by the NETs.
9. Continue to co-culture for an additional two days and take continuous photos of the field every 6 min under normal light and fluorescence, which detects MKN45 cells and NETs, respectively.
10. Superimpose the images at each time point and construct time-lapse videos using Image Viewer software.