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

1. Staining of Tumor Cells with Red Fluorescent Cell Linker Dye

1. Prepare human cancer cell lines MKN45, NUGC, and OCUM-1.
2. Wash 1 x 10⁷  cells with PBS + 0.02% EDTA in a 15 mL tube and centrifuge at 270 x g for 7 min at RT.
3. Add 1 mL of solution for dye staining to the pellets and pipette gently.
4. Dissolve 4 µL of Red Fluorescent Cell Linker dye in 1 mL of solution for staining and mix with the cell suspension from step 1.2.
5. Incubate the pellets for 4 min at RT.
6. Add 4 mL of DMEM (10% FBS) to stop the staining reaction.
7. Centrifuge 270 x g for 7 min and discard the supernatant.
8. Repeat steps 1.6 and 1.7 twice.
9. Check with a fluorescence microscope (excitation = 551 nm, emitting = 567 nm) that the tumor cells are stained red.

2. Analysis of Tumor Cell Adhesion to NETs

1. Culture low-density neutrophil cells in a 6-well poly-L-lysine coated plate (6 cm in diameter) for 2 h at 37 ˚C in 5% CO₂, which enables the production of neutrophil extracellular traps or NETs
2. Transfer the red fluorescent-stained tumor cells (1 x 10⁶) in 1 mL of RPMI 1640 supplemented with 0.1% BSA.
3. Add the tumor cells to the LDN culture that produced the NETs.
4. Incubate for 5 min at 37 ˚C, which enables the tumor cells to contact the NETs.
   NOTE: Long incubation induces tumor cell adhesion to LDN or to the plates.
5. Remove the medium and gently wash the wells by adding 2 mL of prewarmed media (0.1% BSA + RPMI 1640) and swirling the dish.
6. Repeat the washing procedure in step 2.5 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 green fluorescent dye for staining the nucleus and chromosomes at a final concentration of 5 µg/mL for visualization of the NETs.
8. Observe the NETs and attached tumor cells using the appropriate filters (green, excitation = 504 nm, emitting = 523 nm; red, excitation = 551 nm, emitting = 567 nm).
9. Merge the figures to show the tumor cells that are trapped by the NETs.
   NOTE: In some experiments, DNA degradation enzyme was added to the LDN culture (final concentration of 100 U/mL) 5 min before co-incubation for 5 min.