Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

To isolate neutrophils from a tumor-bearing mouse, begin by diluting 1 milliliter of blood, obtained by cardiac puncture, in PBS containing 0.5% BSA to a final volume of 6 milliliters.

Next, add 3 milliliters of sterile-filtered 1.119 grams per milliliter sucrose to the bottom of a 15-milliliter conical polypropylene centrifuge tube. Then, tilting the tube, slowly and carefully layer 3 milliliters of sterile-filtered 1.077 grams per milliliter sucrose on top of the 1.119 grams per milliliter of sucrose followed by the 6 milliliters of diluted blood.

Separate the cells on the sucrose gradient by centrifugation for 30 minutes at 700 g's at room temperature with the break-off.

At the end of the separation, most of the erythrocytes will be at the bottom of the tube. The high-density neutrophils will be around the 3-milliliter mark in the white-to-red ring at the interface between the sucrose layers while the low-density leukocytes will be in the white ring at the interface between the 1.077 grams per milliliter layer, and the BSA-containing PBS around the 6-milliliter mark.

Aspirate the PBS-BSA layer until 5 millimeters above the low-density cell layer. Then, using a 1 1-milliliter pipette tip, remove the low-density cells by slow suction while slowly swirling the cells.

Dispense the cells into 30 milliliters of PBS with 0.5% BSA, then, transfer the high-density neutrophils into a separate tube with 30 milliliters of PBS plus BSA in the same way. Spin down both sets of cells.

Then, resuspend the pellets in 36 milliliters of sterile HPLC-grade water. After 30 seconds, restore the isotonicity of the cells with 9 milliliters of 5x PBS supplemented with 2.5% BSA, and spin down the cells again. Resuspend the cells in 10 milliliters of PBS-BSA and determine the number of viable cells by trypan blue exclusion.

Then, after another centrifugation, resuspend the neutrophils in incubation medium at the appropriate final cell density.