How to Prepare a Polymorphonuclear Cell Fraction from Whole Blood using Ammonium Chloride Lysis

Whole blood is composed of plasma, red blood cells (RBCs or erythrocytes), platelets, and nucleated white blood cells, also referred to as leukocytes. The leukocytes can be further categorized into polymorphonuclear cells, also known as granulocytes, and mononuclear cells. The isolation of polymorphonuclear cells from whole blood is essential for studying neutrophils, eosinophils, basophils, and mast cells. This protocol describes how to isolate polymorphonuclear cells from whole blood using density gradient centrifugation followed by treatment with Ammonium Chloride Solution for RBC lysis. To learn more about cell isolation from blood, see our guide Everything You Need to Know About Cell Isolation.


Materials

*Certain products are only available in select territories. Please contact your local Sales representative or the Product & Scientific Support team at techsupport@stemcell.com for further information.


Protocol

  1. Add Lymphoprep™ to a centrifuge tube. Refer to Table 1 for recommended volumes for various tube sizes.

    Table 1. Recommended Volumes and Tube Sizes

    Blood (mL)
    PBS + 2% FBS (mL)
    Lymphoprep™ (mL)
    Tube Size (mL)
    1
    1
    1.5
    5
    5
    5
    3
    14
    3
    3
    3
    14
    4
    4
    4
    14
    5
    5
    10
    50
    10
    10
    15
    50
    15
    15
    15
    50

  2. Dilute whole blood with an equal amount of Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum (PBS + 2% FBS), or other suitable culture medium.
  3. Layer diluted blood on top of Lymphoprep™, being careful to minimize mixing of blood with Lymphoprep™.
  4. Centrifuge at 800 x g for 20 minutes at room temperature (15 - 25°C) with the brake off.
  5. Remove and discard the plasma layer, the band of mononuclear cells, and Lymphoprep™, leaving the red blood cell (RBC)/granulocyte pellet intact.

    Optional: At this point the pellet may be transferred to a new tube in order to avoid contamination by remaining mononuclear cells left in the tube.
  6. Add Ammonium Chloride Solution to the RBC pellet, completely filling the tube. Incubate on ice for 10 minutes.
  7. Centrifuge at 500 x g for 10 minutes at room temperature with the brake set to low.
  8. Remove and discard the supernatant. Add PBS + 2% FBS to resuspend the pellet, then centrifuge at 120 x g for 10 minutes at room temperature with the brake off.
  9. Remove and discard the supernatant. Resuspend the resulting polymorphonuclear cells in appropriate medium (e.g. PBS + 2% FBS).
Cell isolation kits are available for the positive selection, depletion and negative isolation of total granulocytes, and for the negative selection of neutrophils, eosinophils, and basophils from whole blood or the polymorphonuclear cell fraction. Find more information on our products for the isolation and characterization of polymorphonuclear cell subsets.

Lymphoprep™ is a trademark of Alere Technologies.
  • Document #PR00016
  • Version 1.0.0
  • May 2020


  • Related Resources

    Choosing a Cell Separation Method that Meets Your Research Needs

    Learn more about the cell separation methods outlined above to choose the best method for your application.

    Compare Methods >

    Isolation of CD4+ T cells

    Evaluation Criteria for Cell Separation Methods

    There are many parameters that you can consider when choosing the right cell isolation method for your research.

    View More Parameters >