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

Protocolo

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.

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Materiales

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