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.