Preparing a Polymorphonuclear Cell Fraction from Whole Blood using Ammonium Chloride Lysis

  1. Collect whole blood in a blood collection tube containing heparin or another anticoagulant.
  2. Add Lymphoprep™ to the tube. For appropriate volumes, please see the product information sheet.
  3. Dilute the whole blood with an equal amount of Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum (PBS + 2% FBS; Catalog #07905), or other suitable culture medium.
  4. Layer diluted blood on top of Lymphoprep™, being careful to minimize mixing of blood with Lymphoprep™.
  5. Centrifuge for 20 minutes at 800 x g at room temperature (15-25oC), with the brake off.
  6. Remove and discard the plasma layer, the band of mononuclear cells and the 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.
  7. Resuspend the red cell pellet in Ammonium Chloride Solution (Catalog #07800 / 07850) for lysis of RBCs. Completely fill the tube with RBC lysis solution and incubate on ice for 10 minutes.
  8. Centrifuge for 10 minutes at 500 x g at room temperature, with the brake set to low.
  9. Discard the supernatant and wash the pellet in PBS + 2% FBS to remove platelets, centrifuging for 10 minutes at 120 x g at room temperature, with the brake off.
10. Resuspend the resultant polymorphonuclear cells (PMNCs) in appropriate medium (e.g. PBS + 2% FBS).

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 PMNC fraction. on our products for the isolation and characterization of PMNC subsets.

Lymphoprep™ is a trademark of Axis-Shield.

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