How to Remove 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 samples, immunodensity cell separation can be used to support Lymphoprep™- or Ficoll™-based elimination of granulocytes using density gradient centrifugation.

This protocol describes how to remove granulocytes by immunodensity cell separation using RosetteSep® Human Granulocyte Depletion Cocktail. Additionally, a second option is provided in which a SepMate™ tube is used to harvest isolated mononuclear cells with a simple pour. Performing immunodensity cell separation before density gradient centrifugation can result in a blood sample containing < 1% granulocytes, compared to 20% granulocytes with density gradient centrifugation alone.


Option 1: Density Gradient Centrifugation Using a Non-SepMate™ Tube

Materials


Protocol

Before You Begin: Ensure that the whole blood sample, PBS + 2% FBS, Lymphoprep™, and centrifuge are all at room temperature (15 - 25ºC).

  1. Add RosetteSep® Human Granulocyte Depletion Cocktail at 50 µL/mL of whole blood and incubate at room temperature for 20 minutes.
  2. Dilute whole blood with an equal volume of PBS + 2% FBS and mix gently.
  3. Layer the diluted sample on top of the Lymphoprep™. Be careful to minimize mixing of the density gradient medium and the sample.
  4. Centrifuge at 1200 x g for 20 minutes at room temperature (with the brake off).
  5. Remove the enriched cells from the Lymphoprep™:plasma interface.
  6. Wash enriched cells with PBS + 2% FBS.
  7. Repeat wash step.


Option 2: Density Gradient Centrifugation Using a SepMate™ Tube

Materials


Protocol

Before You Begin: Ensure that the whole blood sample, RosetteSep® Human Granulocyte Depletion Cocktail, PBS + 2% FBS, Lymphoprep™, and centrifuge are all at room temperature (15 - 25ºC).

  1. Add RosetteSep® Human Granulocyte Depletion Cocktail at 50 µL/mL to whole blood and incubate at room temperature for 10 minutes.
  2. Dilute whole blood with an equal volume of PBS + 2% FBS and mix gently.
  3. Add Lymphoprep™ to the SepMate™ tube through the hole in the insert.
  4. Pipette the diluted sample down the side of the SepMate™ tube.
  5. Centrifuge at 1200 x g for 20 minutes at room temperature (with brake on).
  6. Pour off the top layer, which contains the enriched mononuclear cells, into a new tube. Do not hold the SepMate™ tube in the inverted position for longer than 2 seconds.
  7. Wash enriched cells with PBS + 2% FBS.
  8. Repeat wash step.


Note: To minimize platelet contamination, remove and discard the top third of the plasma layer before collecting the cells at the density gradient medium:plasma interface. Platelets may also be removed by including an extra wash with centrifugation at 120 x g for 10 minutes at room temperature with the brake OFF after step 8.
  • Document #PR00011
  • Version 1.0.0
  • Apr 2020


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