Isolation and Characterization of Neutrophils with Anti-Tumor Properties

The overall goal of this procedure is to study neutrophil function in cancer. This is accomplished by first obtaining the peripheral blood. In the second step, the neutrophils are purified on a density gradient followed by overnight co-culture.

With luciferase labeled tumor cells, the luciferase activity of the cells is then quantified. Ultimately, the amount of the luciferase in each co-culture can be inversely correlated to the cytotoxicity of the neutrophils within each co-culture. The main advantage of this technique over existing methods like antibody-based neutrophil separation and other methods for evaluating cell viability are that this procedure minimizes the non-specific activation of neutrophils and allows the measurement of cell viability without the use of radioactive labels.

Demonstrating this procedure will be, it has a give a graduate student in my laboratory, and Dr.Simoni, a postdoctoral fellow in my laboratory To isolate neutrophils from a tumor bearing mouse begin by diluting one milliliter of blood obtained by cardiac puncture in PBS containing 0.5%BSA to a final volume of six milliliters. Next, add three 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 three milliliters of sterile filtered 1.077 grams per milliliter sucrose on top of the 1.119 grams per milliliter of sucrose, followed by the six milliliters of diluted blood. Separate the cells on the sucrose gradient by centrifugation for 30 minutes at 700 Gs 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 three 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 six milliliter mark aspirate the P-B-S-B-S-A layer until five millimeters above the low density cell layer. Then using a one 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 nine milliliters of five XPBS supplemented with 2.5%BSA and spin down the cells. Again, re suspend the cells in 10 milliliters of P-B-S-B-S-A and determine the number of viable cells by trian blue exclusion. Then after another centrifugation resus suspend the neutrophils in incubation medium at the appropriate final cell density.

To isolate the circulating neutrophils from a cancer patient. Begin by mixing 10 milliliters of heparinized human blood with an equal volume of 3%DExT strand T 500 in saline for a 30 minute incubation at room temperature to sediment the erythrocytes when the red blood cells have settled, slowly layer the leukocyte rich supernatant over 10 milliliters of 1.077 grams per milliliter sucrose, and separate the cells by centrifugation. As just demonstrated the human high density neutrophils will be present in the palate while the low density neutrophils will co purify with the monocytes and lymphocytes at the interface between the 1.077 grams per milliliter sucrose layer and the plasma.

After collecting the neutrophils as just demonstrated, dilute the cells in 10 milliliters of 0.2%sodium chloride for 30 seconds to ly the contaminating erythrocytes. Then add 10 milliliters of 1.6%sodium chloride to restore isotonicity to the cells and invert the tube once to mix. Spin down the cells and wash the pellet three times in 20 milliliters of HBSS.

Then count the cells and resuspend them in RPMI 1640, supplemented with 2%FBS at the appropriate cell density. To isolate thi glyco or Xan elicited neutrophils. Use a tuberculin syringe with a 23 gauge one inch needle to intraperitoneal.

Inject one milliliter of the appropriate solution into the mouse's abdomen. Four to 24 hours later, inject five milliliters of sterile PBS into the peritoneum of the euthanized animal with a five milliliter syringe equipped with a 25 gauge five eights inch needle. Gently massage the peritoneum, then redraw the fluid back to the syringe to collect the peritoneal exudate cells.

Wash the cells in RPMI 1640, medium supplemented with 10%FBS. Then to remove the macrophages, incubate one 10 to the six cells per milliliter in a tissue culture grade culture dish containing medium supplemented with FBS for two hours at 37 degrees Celsius. To stain the isolated neutrophils, resus suspend one times 10 to the fifth of the cells in 50 microliter aliquots of PBS and spin the cell suspensions in a thin layer cell preparation adapter at 150 Gs.After five minutes, separate the pre-labeled glass slides from the adapter, fix and stain the cells.

Next, wash the slides 15 times in tap water. Let the slides air dry again, and then inspect the cells under a light microscope to assess the anti-tumor activity of the neutrophils. Dilute five times 10 to the fourth luciferase labeled tumor cells per milliliter in optimized reduced serum medium, supplemented with 0.5%FBS.

Then seed five times 10 to the third of the tumor cells in 100 microliters of optimized reduced serum medium containing 0.5%FBS in each well of a white 96 well flat bottom tissue culture plate. Four hours later at one times 10 to the fifth neutrophils in 50 microliters of optimized reduced serum medium containing 0.5%FBS to each well for an overnight incubation. The following morning, gently aspirate the supernatant and wash each well with 200 microliters of PBS.

Then aspirate the PBS and add 50 microliters of passive lysis buffer to each well. Next incubate the plate covered with foil on an orbital shaker at 150 RPM at room temperature. After 20 minutes, load the plate in luminescence plate reader.

Inject 50 microliters of luciferase assay solution into each well and read the chem luminescence for 10 seconds per well. Finally use the formula to calculate the percentage of tumor lysis to test the extent of neutrophil cytotoxicity in this representative experiment one times 10 to the fifth. Neutrophils were co cultured with luciferase expressing four T one target cells as just demonstrated in co cultures with neutrophils purified from tumor free mice.

Very little killing of the tumor cells was observed. Neutrophils purified from tumor bearing mice, however, exhibited a significant level of cytotoxicity to test the immunosuppressive properties of the low and high density neutrophils. The number of CD eight positive splenic T cells after stimulation with anti CD three or anti CD three plus low or high density neutrophils was assessed after anti CD three activation.

A dramatic increase in the CD eight positive cells was observed. Co-culture with low density neutrophils, however, induced a noticeable reduction in the CD eight positive cells, an effect that was not observed in the presence of the high density neutrophils. The extent of CFSE retention was also evaluated as an indicator of proliferation.

Note the left word shift in the anti CD three treated cells and the anti CD three treated cells cultured in the presence of high density neutrophils demonstrating in both cases an increase in the proliferation of CD eight positive cells. Following this procedure. Other methods like measuring oxidative bursts, phagocytosis, or migration can be performed to answer additional questions like, how is neutrophil function affected under different pathological scenarios?