Peritoneal Low-density Neutrophil Isolation: A Technique to Obtain Low-density Neutrophils from Peritoneal Lavage Fluid Using Magnetic Activated Cell Sorting

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

1. Sample acquisition
   1. 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 gastrointestinal malignancy.
      NOTE: Samples were obtained from patients who underwent gastrectomy, colectomy or esophagectomy without bia....

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 separat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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