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

Published: April 30, 2023

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 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.

Divulgations

The authors have nothing to disclose.

Materials

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

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Time-lapse Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Analyze Post-adhesion Interaction between Cancer Cells and NETs. J. Vis. Exp. (Pending Publication), e20416, doi: (2023).

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