Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Neutrophil Isolation

1. Isolation of cytotoxic neutrophils from blood of tumor-bearing mice.
   1. Dilute 1 ml of blood drawn from a tumor-bearing mouse in PBS containing 0.5% (w/v) bovine serum albumin (BSA) to a final volume of 6 ml.
   2. Fractionation of diluted blood on a freshly prepared discontinuous sucrose gradient:
      1. Add 3 ml of sterile-filtered sucrose 1.119 g/ml to the bottom of a 15 ml conical polypropylene centrifuge tube.
      2. Slowly and carefully, layer 3 ml of sterile-filtered sucrose 1.077 g/ml on top of the 1.119 g/ml layer. Thereafter, add slowly and carefully the 6 ml of diluted blood on top of the 1.077 g/ml layer (Figure 1A). It is recommended to hold the tube tilted and add the different components by a slow, but continuous flow, keeping the mouth of the pipette towards the lower wall of the tube, such that no turbulence is formed.
   3. Centrifuge the tube containing the diluted blood on the sucrose gradient at 700 x g for 30 min at RT without brake.
   4. Carefully remove the tube from the centrifuge without causing any turbulence. Most of the erythrocytes will be at the bottom of the tube. High-density neutrophils (HDN) are found as a white-to-red ring at the interface between the 1.119 g/ml and the 1.077 g/ml layers (around the 3 ml mark), while the low-density leukocytes are found in a white ring at the interface between the 1.077g/ml layer and the BSA-containing PBS (around the 6 ml mark, see Figure 1B).
   5. Aspirate the PBS + 0.5% BSA until reaching 5 mm above the low-density cell layer. Pipette out the low-density cells by slow suction into a 1 ml tip through slowly swirling around the cells. Transfer the cells into 30 ml of PBS with 0.5% BSA.
   6. Aspirate the upper layer in the same gradient tube until reaching 5 mm above the high-density cell band. Pipette out the high-density cells, which are mostly high-density neutrophils, and transfer the cells into 30 ml of PBS containing 0.5% BSA.
   7. Centrifuge the cells at 400 x g for 10 min at RT.
   8. Aspirate the supernatant and lyse erythrocytes by resuspending the cells in 36 ml sterile HPLC grade water for 30 sec. Isotonicity should be restored by adding 9 ml of a 5x concentrated PBS supplemented with 2.5% (w/v) BSA.
   9. Centrifuge the cells at 400 x g for 10 min at RT.
   10. Aspirate the supernatant, and resuspend the cells in PBS-BSA. Count the number of neutrophils in a hemocytometer and use trypan blue to distinguish between live and dead cells.
   11. Centrifuge the cells at 400 x g for 10 min at RT and resuspend the neutrophils in desired incubation medium to final cell density. Use the neutrophils immediately.
   12. Then, after another centrifugation, resuspend the neutrophils in an incubation medium to final cell density. Use the neutrophils immediately.