Why do neutrophils die during hemolysis?

Dear Rosemari,

Neutrophils are very sensitive!

Article Neutrophil Isolation Protocol

. 2008 Jul 23;(17):745. doi: 10.3791/745 📷J Vis Exp

Neutrophil Isolation Protocol

Bring all reagents to room temperature.

Collect 5.0 ml of neutrophil isolation media in centrifuge tube. Carefully layer 5.0 ml of blood over the separation media. Perform this step slowly and carefully, and with the pipette tip close to the surface of the media to avoid mixing the blood and the media.

Centrifuge at 500 RCF for 35 min at 20-25°C. The blood should separate out into 6 distinct bands: plasma, monocytes, isolation media, neutrophils, more isolation media, and the red blood cell pellet (Figure 1). If these bands are not clear, the separation process was not clean and will need to be repeated.

Carefully remove the top three layers (plasma, monocytes, and isolation media) using a pipette. Dispose of these layers.

Carefully pipette the layer of neutrophils and all of the isolation media beneath the neutrophils. Place the solution into a clean centrifuge tube.

Dilute the neutrophil solution to 10 ml with HBSS without Ca2+/Mg2+. Invert the tube a few times to suspend the cells.

Centrifuge the neutrophil solution at 350 RCF for 10 minutes. A red pellet should be present at the bottom of the tube, containing neutrophils and residual red blood cells (RBCs). Remove the supernatant with a pipette carefully so that the pellet is not disturbed.

To lyse the residual RBCs, add 2 ml Red Cell Lysis Buffer to the tube. To resuspend the pellet, vortex the vial at a setting of 3-4. Avoid increasing the vortex setting above 4, since this may cause the neutrophils to activate. It may be necessary to vortex for several seconds, or to "pulse" the vortex to dissolve the pellet.

Centrifuge the tube at 250 RCF for 5 min. Remove the supernatant with pipette. Repeat the lysing process if required.

Add 500 μl HBSS without Ca2+/Mg2+ to each tube. Again, vortex to resuspend the pellet at a setting of 3-4. Dilute to 10 ml with HBSS without Ca2+/Mg2+.

Centrifuge the tubes at 250 RCF for 5 min. Aspirate the supernatant and discard.

Resuspend the pellet in 250 μl HBSS/HSA Solution (2% HSA). Cells may then be counted and adjusted to desired concentration.

Best regards,

Thomas

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