Peritoneal Low-density Neutrophil Isolation: A Technique to Obtain Low-density Neutrophils from Peritoneal Lavage Fluid Using Magnetic Activated Cell Sorting
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 describe the procedure for isolating low-density neutrophils, or LDNs, from the peritoneal lavage fluid of a postoperative human patient. The LDNs thus obtained can be used in further downstream experiments to understand their role in tumor growth and progression.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Isolation of LDN from Abdominal Cavity Lavages Sample acquisition Infuse 1,000 mL of normal sterile saline directly into the abdominal cavity before wound closure in patients who have undergone abdominal surgery due to gast…
Representative Results
Figure 1: NET formation after the culture of peritoneal LDN. (A) After density gradient centrifugation of postoperative lavage fluid, cells recovered from the intermediate layer are shown. (B) Then, CD66b(+) LDN were separated from CD66b(-) mononuclear cells and cultured on poly-L-lysine coated 6-well plates, followed by nuclear staining dye additio…
Divulgations
The authors have nothing to disclose.
Materials
| Ficoll-Paque PLUS | GE Healthcare, SWEDEN | 17-1440-02 | |
| StraightFrom™ Whole Blood CD66b MicroBeads | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-104-913 | |
| Fc block | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-059-901 | |
| MACS Rinsing Solution | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-091-222 | |
| 0.5mol/l-EDTA Solution (pH 8.0) | Nacalai tesque, Japan | 06894-14 | |
| MACS BSA Stock Solution | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-091-376 | |
| LS Columns | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-041-306 | |
| MACS Magnetic Separator | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-042-501 |
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Citer Cet Article
Peritoneal Low-density Neutrophil Isolation: A Technique to Obtain Low-density Neutrophils from Peritoneal Lavage Fluid Using Magnetic Activated Cell Sorting. J. Vis. Exp. (Pending Publication), e20414, doi: (2023).