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

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 bias based on age or sex. Saline was transferred into a container and poured into the whole abdomen within one minute. This is routinely performed as a postoperative peritoneal washing without significant effects on patients.
   2. Lavage the abdominal cavity extensively for at least 1 min.
      NOTE: It is recommended that the infused fluid is slowly stirred with the surgeon's hands so that the samples are uniform.
   3. Recover 200 mL of lavage fluid with four 50 mL syringes.
      NOTE: Sometimes, a rubber connector is used to take up the fluids.
2. Perform purification of peritoneal LDN using granulocyte specific mAb, CD66b
   NOTE: Because the intermediate layer after density gradient centrifugation contains many mononuclear cells, a positive selection of polymorph neutrophils using CD66b mAb was performed.
   1. Transfer the peritoneal lavage fluid to a 50 mL tube.
      NOTE: In this step, pass the fluids through a 100 µm nylon filter to remove impurities.
   2. Centrifuge the peritoneal fluid at 270 x g for 7 min at RT.
   3. Resuspend the pellets in 5 mL of PBS with 0.02% EDTA.
   4. Carefully overlay the 5 mL of cell suspension on a 3 mL density gradient solution.
   5. Centrifuge at 1,700 x g for 15 min at RT without any breaks.
   6. Harvest around 2 to 3 mL of a solution containing the intermediate layers (Figure 1A) using a pipette and mix it with 10 mL of PBS with 0.02% EDTA.
   7. Centrifuge the peritoneal fluid at 400 x g for 7 min at RT and discard the supernatant.
   8. Add another 10 mL of PBS with 0.02% EDTA and centrifuge at 270 x g for 7 min at RT. Discard the supernatant.
   9. Dissolve the cell pellet (1 x 10⁷) in 60 µL of buffer for the magnetic cell separation kit.
   10. Add 20 µL of Fc block to the pellets and incubate for 10 min at 4 °C.
   11. Add 20 µL of the anti-CD66b conjugated with microbeads and incubate for an additional 10 min at 4 °C.
   12. Wash and resuspend the pellets in 500 µL of MACS buffer.
   13. Apply the cell suspension to a magnetic column in the magnetic field of a suitable magnetic separator and collect the flow-through containing CD66b(-) cells by washing the column with 15 mL of buffer.
   14. Remove the column from the magnetic separator and immediately flush out the magnetically labeled CD66b(+) cells by firmly pushing the plunger into the column.
       NOTE: The CD66b(+) cell population consists mostly of neutrophils as determined by FACS analysis, indicating that >95% of the CD66(+) fraction are positive for CD11b, CD15, and CD16, but negative for CD14.

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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...

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Materials

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