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

Protocole

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.

matériels

Name	Company	Catalog Number	Comments
SYTOX green nucleic acid stain 5mM solution in DMSO	Thermo Fisher Scientific, Waltham, MA, USA	S7020
RPMI1640 Medium	Sigma-Aldrich, St Louis, MO, USA	R8758
Dulbecco’s Modified Eagle Medium-high glucose (DMEM)	Sigma-Aldrich, St Louis, MO, USA	D5796
Dulbecco’s Phosphate Buffered Saline (DPBS)	Sigma-Aldrich, St Louis, MO, USA	D8537
Fetal Bovine Serum, qualified, USDA-approved regions	gibco by life technologies, Mexico	10437-028
Bovine Serum Albumin lyophilized powder, ≥96% (agarose gel electrophoresis)	Sigma-Aldrich, St Louis, MO, USA	A2153
Penicillin Streptomycin	Life Technologies Japan	15140-122
Plasmocin Prophylactic	InvivoGen, San Diego, CA-USA	ant-mpp
Fluorescein stereomicroscope	BX8000, Keyence, Osaka Japan	BZ-X710
Whole view cell observation system	Nikon, Kanagawa, Japan	BioStudio (BS-M10)
MKN45 human gastric cancer cell line	Riken, Tukuba Japan	N/A