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

Low-density neutrophils, or LDNs, are a distinct neutrophil subpopulation found in elevated numbers during pathological conditions like gastrointestinal cancer.  LDNs can be isolated from the peritoneal cavity washes or lavage fluid of patients.

First, filter the lavage fluid to remove tissue debris. Centrifuge the filtrate to obtain a pellet containing blood cells, including LDNs. Resuspend it in a suitable buffer. Layer this suspension over a suitable density gradient medium optimal for LDN isolation.

Centrifuge to separate the different blood components based on their relative densities. The denser RBCs form the bottom layer, while the least dense plasma fraction forms the topmost layer. The less-dense LDNs occupy the intermediate layer along with other mononuclear cells.

Transfer the intermediate layer into a fresh tube containing a blocking reagent. The reagent components block non-specific binding sites on all non-target cells' surfaces and increase the specificity for LDN during subsequent labeling.

Add antibody-conjugated microbeads targeting a specific cell surface molecule overexpressed on LDNs. Incubate for the antibody-bead complexes to bind to their target molecules on LDNs.

Pass the incubated suspension through a column placed in a magnetic field. Under the magnetic influence, all LDN-microbead complexes adhere to the column wall while unlabeled cells pass through.

Remove the column. Flush the contents using a suitable buffer to elute and collect the LDN-containing fraction.

To acquire the neutrophils, first, infuse 1 liter of normal sterile saline immediately before wound closure directly into the abdominal cavity of a patient who has just undergone abdominal surgery due to gastrointestinal malignancy, and lavage the abdominal cavity extensively for at least 1 minute. Then, stir the saline within the cavity and recover 200 milliliters of lavage fluid with four 50-milliliter syringes.

In the lab, transfer the peritoneal lavage fluid through a 100-micrometer nylon filter into individual 50-milliliter tubes for centrifugation. Resuspend the pellets in 5 milliliters of PBS supplemented with 0.02% EDTA and carefully overlay each cell suspension onto 3 milliliters of density gradient solution. After density gradient separation, harvest 2 to 3 milliliters of the intermediate layer from each tube and wash the peritoneal fluid samples in 10 milliliters of fresh PBS plus EDTA per tube. Pour the pellets in 10 milliliters of fresh PBS plus EDTA for an additional wash and dilute the cells to a 1 times 10 to the seventh cells per 60 micrometers of magnetic activated cell sorting, or MACS, buffer concentration.

Block any non-specific binding with 20 microliters of Fc block for 10 minutes at 4 degrees Celsius, followed by the addition of 20 microliters of anti-CD66b magnetic beads. After 10 minutes at 4 degrees Celsius, wash and resuspend the cells in 500 microliters of MACS buffer and add the cells to an appropriately sized magnetic column within the magnetic field of a suitable magnetic separator. When all of the CD66b negative cells have run through the column, add 15 milliliters of fresh MACS buffer to the column reservoir and transfer the column from the magnetic separator into a new conical tube. Then, immediately plunge the column to flush the magnetically labeled CD66b positive into the tube.

• Low-density neutrophils (LDNs) - A distinct neutrophil subpopulation increased in pathological conditions.
• Ficoll-Paque Plus protocol - Method for isolating neutrophils using density gradient separation.
• Peritoneal lavage procedure - Technique for washing and collecting fluid from the peritoneal cavity.
• Magnetic cell sorting (Miltenyi Biotec) - Technique to selectively isolate specific cells using magnetic microbeads.
• CD66b - A surface molecule overexpressed on LDNs, useful for their identification and isolation.

• Intrduce LDNs – Subtype of neutrophil prevalent in diseases like cancer (e.g., low density neutrophils).
• Outline Ficoll-Paque Plus protocol – Method for cell separation based on density (e.g., Ficoll-Paque Plus protocol).
• Explain Peritoneal lavage procedure – Technique to collect cells from the peritoneal cavity (e.g., peritoneal lavage procedure).
• Connect to Magnetic cell Sorting – Isolation of LDNs using magnetic microbeads and specific markers (e.g., CD66b).

• What are low-density neutrophils and how to identify them using peritoneal lavage procedure?
• How does Ficoll-Paque Plus protocol help in separating LDNs?
• How does magnetic cell sorting with CD66b microbeads isolate LDNs?

• Practical Applications – Isolation of LDNs for clinical and research purposes (e.g., Ficoll-Paque Plus protocol).
• Industry Impact – Advances in cell isolation techniques benefitting biomedical sector (e.g., magnetic cell sorting).
• Societal Importance – Improved understanding and treatment of certain diseases (e.g., cancer).
• Link to Scientific Advancements – Automatic isolation of specific cells aiding in research studies.