Removing Granulocytes from Old Blood Samples

Granulocytes change density as blood samples age. This results in granulocyte contamination of mononuclear cells when processing older blood samples (>24 hours post collection) using a density gradient medium. For effective granulocyte depletion in older human whole peripheral blood, we recommend using RosetteSep® Human Granulocyte Depletion Cocktail (Catalog #15624) to assist Lymphoprep™ or Ficoll™ in eliminating granulocytes. Use a SepMate™ tube to harvest the isolated mononuclear cells with a simple pour.

A simple step before Lymphoprep™ or another density gradient medium:

Add RosetteSep® Human Granulocyte Depletion Cocktail (Catalog #15624) at 50 µL/mL of whole blood and incubate for 20 minutes at room temperature. Dilute whole blood with an equal volume of PBS + 2% FBS and mix gently. Then follow the procedure as outlined in the table below:

Lymphoprep™

Lymphoprep™ with SepMate™

Layer the diluted sample on top of the Lymphoprep™ (Catalog #07801)

Pipette the diluted sample down the side of the SepMate™ tube

Centrifuge at 1200 x g for 20 minutes at room temperature (with the brake off)

Centrifuge at 1200 x g for 20 minutes at room temperature (with brake on)

Remove the enriched cells from the Lymphoprep™: plasma interface

Pour off the top layer, which contains the enriched MNCs, into a new tube. Do not hold the SepMate™ tube in the inverted position for longer than 2 seconds.

Wash enriched cells with PBS + 2% FBS (Catalog #07905)

Wash enriched cells with PBS + 2% FBS (Catalog #07905)

Repeat wash step

Repeat wash step


Results:

  • Start: 66% Granulocytes
  • Lymphoprep™ alone: 20% Granulocytes
  • RosetteSep® Human Granulocyte Depletion Cocktail: <1% Granulocytes

For further assistance and information, please contact techsupport@stemcell.com