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

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.