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

Abstract

Source: Kanamaru, R. et al. Neutrophil Extracellular Traps Generated by Low-density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment. J. Vis. Exp. (2018)

In this video, we analyze the post-adhesion behavior of cancer cells trapped in neutrophil extracellular traps (NETs) by co-culturing low-density neutrophils and gastric cancer cells. The time-lapse video analysis helps monitor various cellular events and study the role of NETs in pathological conditions.

Protocol

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 addin....

Materials

List of materials used in this article

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