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

Overview

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

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.

Materials

Name	Company	Catalog Number	Comments
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