EasySep™小鼠TIL（CD45）正选试剂盒
Removing Granulocytes from Old Blood Samples
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 (Catalog #15624) to assist 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 ) 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 #) |
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 #) |
Wash enriched cells with PBS + 2% FBS (Catalog #) |
|
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
