Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

To isolate neutrophil subpopulations, including cytotoxic high-density neutrophils, HDNs, and low-density neutrophils, LDNs, from the blood of a tumor-bearing mouse, take a tube containing the mouse blood.

Dilute with buffer containing bovine serum albumin, BSA, proteins, which maintain the osmotic balance and stabilize the blood cells. Layer an appropriately diluted blood volume on a discontinuous sucrose gradient comprising two non-overlapping sucrose layers with differing densities. 

During centrifugation, the cells migrate to different regions in the gradient based on their densities relative to the gradient medium. The majority of the high-density erythrocytes sediment to the tube bottom.

HDNs with granulocytes form a white-red ring at the interface between the sucrose layers, while the LDNs and mononuclear cells form a white ring at the interface between the low-density sucrose layer and the BSA-containing buffer layer.

Remove the top BSA-containing buffer layer. Transfer the low-density cell layer to a tube with BSA-containing buffer. Remove the low-density sucrose layer. Transfer the high-density cell layer to a tube with BSA-containing buffer.

Centrifuge the tubes. Resuspend the cell pellets in sterile water to lyse the erythrocytes. Add a buffer with a higher BSA concentration to restore the cell isotonicity. Centrifuge the tubes. Discard the supernatant containing the lysed erythrocytes.

Resuspend the cells containing LDNs and HDNs in BSA-containing buffer for further analysis.

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.