Overview
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
- 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.
- Add the green-fluorescent dye for staining the nucleus and chromosomes to visualize NETs at the final concentration of 5 µM.
- After 1 min of incubation, remove the media and add 2 mL of 0.1% BSA + RPMI 1640.
- Remove the media and add unstained 1 x 106 MKN45 cells suspended in 0.1% BSA + RPMI 1640. Incubate for 5 min at RT.
- 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.
- 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. - Add DMEM with 10% FCS supplemented with 100 u/mL Penicillin and 100 µg/mL streptomycin.
- 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.
- 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.
- Superimpose the images at each time point and construct time-lapse videos using Image Viewer software.
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Materials
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 |