Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Observe Adhesive 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)

This video demonstrates the fluorescent staining of neutrophil extracellular traps (NETs), the extracellular DNA components expelled by neutrophils. This technique enables the visualization of adhesive interaction between cancer cells and NETs.

Protocol

1. Staining of Tumor Cells with Red Fluorescent Cell Linker Dye Prepare human cancer cell lines MKN45, NUGC, and OCUM-1. 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. Add 1 mL of solution for dye staining to the pellets and pipette gently. 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</stro…

Materials

SYTOX green nucleic acid stain 5 mM solution in DMSO	Thermo Fisher Scientific, Waltham, MA, USA	S7020
PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling	Sigma-Aldrich, St Louis, MO, USA	P9691
RPMI1640 Medium	Sigma-Aldrich, St Louis, MO, USA	R8758
Bovine Serum Albumin lyophilized powder, ≥96% (agarose gel electrophoresis)	Sigma-Aldrich, St Louis, MO, USA	A2153
Poly-L-Lysine-Coated MICROPLATE 6Well	IWAKI, Japan	4810-040
Fluorescein stereomicroscope	BX8000, Keyence, Osaka Japan	BZ-X710
MKN45 human gastric cancer cell line	Riken, Tukuba Japan	N/A
NUGC-4 human gastric cancer cell line	Riken, Tukuba Japan	N/A
OCUM-1 human gastric cancer cell line	Osaka City University, Japan	N/A	Gift from Dr. M. Yashiro
DNase I	Worthington, Lakewood NJ	LS002138
0.5 mol/l-EDTA Solution (pH 8.0)	Nacalai tesque, Japan	06894-14
Dulbecco’s Modified Eagle Medium-high glucose (DMEM)	Sigma-Aldrich, St Louis, MO, USA	D5796