Jul 03, 2019

Public workspaceIsolation of Mononuclear Cells from Whole Blood by Density Gradient Centrifugation

PLOS One
DOI
dx.doi.org/10.17504/protocols.io.4v3gw8n
  • 1STEMCELL Technologies
  • STEMCELL Technologies
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DOI: dx.doi.org/10.17504/protocols.io.4v3gw8n
External link: https://www.stemcell.com/how-to-isolate-mononuclear-cells-from-whole-blood-by-density-gradient-centrifugation.html?utm_source=protocolsio&utm_medium=referral
Protocol Citation: Stemcell Technologies 2019. Isolation of Mononuclear Cells from Whole Blood by Density Gradient Centrifugation. protocols.io https://dx.doi.org/10.17504/protocols.io.4v3gw8n
Manuscript citation:
Fazeli S, Motovali-Bashi M, Peymani M, Hashemi M, Etemadifar M, Nasr-Esfahani MH, Ghaedi K (2020) A compound downregulation of SRRM2 and miR-27a-3p with upregulation of miR-27b-3p in PBMCs of Parkinson’s patients is associated with the early stage onset of disease. PLoS ONE 15(11): e0240855. doi: 10.1371/journal.pone.0240855
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 28, 2019
Last Modified: July 03, 2019
Protocol Integer ID: 25243
Keywords: cell separation, cell isolation, centrifuge, fractionation, peripheral blood, Lymphoprep, Ficoll-Pacque
Abstract
This protocol describes how to isolate mononuclear cells from whole blood using density gradient centrifugation. See how to layer blood over the density medium and learn two different ways of harvesting cells from the blood plasma-density medium interface.
Materials
STEP MATERIALS
ReagentLymphoprep™STEMCELL Technologies Inc.Catalog #07801
Protocol materials
ReagentLymphoprep™STEMCELL Technologies Inc.Catalog #07801
ReagentLymphoprep™STEMCELL Technologies Inc.Catalog #07801
Before start
Ensure all reagents are at room temperature.

The following video demonstrates how to isolate peripheral blood mononuclear cells from whole blood using density gradient centrifugation.
https://www.youtube.com/embed/Cl4sTwKFe9k

Dilute the blood sample at a 1:1 volume ratio with the appropriate medium.
Add a volume of density gradient medium to a fresh tube according to the specifications for that density gradient medium.
ReagentLymphoprep™STEMCELL Technologies Inc.Catalog #07801

Gently layer the diluted blood on top of the density gradient medium. Take care not to mix the two layers.

Centrifuge Centrifigation400 x g , for 30 minutes with the brake off.

Carefully harvest the cells by inserting the pipette directly through the upper plasma layer to the mononuclear cells at the interface.
Note
Alternatively: You can first remove the upper layer and then collect the cells.


Finally, wash the harvested cells twice in the appropriate buffer. The cells are now ready for EasySep™ cell separations or other downstream applications.