Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Observe Adhesive 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)
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…
Divulgaciones
The authors have nothing to disclose.
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 |
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Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Observe Adhesive Interaction Between Cancer Cells and NETs. J. Vis. Exp. (Pending Publication), e20415, doi: (2023).