Dec 17, 2025

Public workspaceViable PBMCs isolation using density-gradient separation

DOI
https://dx.doi.org/10.17504/protocols.io.3byl48ejrvo5/v1
  • Layla Drwesh1,
  • Loris Bandirali2,
  • Julia Fitzgerald1
  • 1Hertie Institute for Clinical Brain Research, University of Tübingen;
  • 2Department of Brain and Behavioural Sciences, University of Pavia, Pavia, Italy.
Julia Fitzgerald
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl48ejrvo5/v1
Protocol Citation: Layla Drwesh, Loris Bandirali, Julia Fitzgerald 2025. Viable PBMCs isolation using density-gradient separation. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl48ejrvo5/v1
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: December 16, 2025
Last Modified: December 17, 2025
Protocol Integer ID: 235141
Keywords: high quality pbmcs from whole blood, gradient separation collection of pbmc, viable pbmcs isolation, alignment across ongoing parkinson, biomarker development, gradient separation collection, high quality pbmc, pbmc, ongoing parkinson, incorporating sepmate tube, stem cell technology, different lab, density gradient method, sepmate tube, blood collection
Funders Acknowledgements:
Michael J Fox Foundation
Grant ID: 026376
Abstract
Collection of PBMCs for biomarker development is an essential part of translational research. Several methods are employed by different labs, including our own. Here we describe in detail, a method (in place in our lab from 2025) to isolate high quality PBMCs from whole blood (EDTA blood) within ~45 minutes from blood collection. We use a density gradient method, incorporating SepMate tubes (Stem Cell Technologies). We would like to share this protocol for the purpose of standardization, comparison and alignment across ongoing Parkinson's disease cohort studies.
Guidelines
Working with blood. Before starting, make sure that all local safety and occupational health rules are followed. For example, vaccinations, safety instructions.
Materials
Guidelines
Except for the centrifugation steps, all sample processing was carried out under a biosafety cabinet in sterile conditions.
Store Lymphoprep, CryoStor CS10, PBS+2%FBS and Erylysis buffer at 4°C.
Before start
Whole peripheral blood samples are collected in EDTA tubes by professional healthcare workers in medical examination rooms.
Prepare 100mL of PBS+2%FBS for two blood vials (~15mL) under the hood.
Prepare 50mL of Erylysis Buffer as follows:
  • 0.1mM EDTA
  • 155mM NH4Cl
  • 10mM KHCO3
  • Adjust the pH to 7.3-7.4 with HCl or NaOH
Bring Lymphoprep, PBS+2%FBS solution, Erylysis buffer, CryoStor CS10 and Mr. Frosty at room temperature (18°C-25°C).
Materials
SepMateTM tubes (50mL) for density gradient separation STEMCELL Technologies Catalog #85450.
Falcon tubes (50mL) Corning Catalog #352070
Falcon Pipettes (25mL) Corning Catalog #357525
Falcon Pipettes (10mL) Corning Catalog #356551
Falcon Pipettes (5mL) Corning Catalog #357543
Cryo. S (2mL) Greiner Bio-One Catalog #122263
LymphoprepTM STEMCELL Catalog #07851/07861
CryoStor CS10 STEMCELL Catalog #07930
1X Dulbecco’s Phosphate Buffer Saline Sigma-Aldrich Catalog #D8537
Fetal Bovine Serum, Premium Thermofisher Catalog #A5670701
Protocol materials
ReagentLymphoprep™ 500 mL STEMCELL Technologies Inc.Catalog #7851
ReagentDulbecco’s Phosphate Buffer Saline Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8537
ReagentFetal Bovine Serum, Premium Gibco - Thermo Fisher ScientificCatalog #A5670701
ReagentCryoStor CS10-100mlSTEMCELL Technologies Inc.Catalog #07930
Reagent50ml Falcon tubesCorningCatalog #352070
ReagentSepMate-50STEMCELL Technologies Inc.Catalog #85450
ReagentDMSOPanreac AppliChemCatalog #A3672
ReagentCRYO.S 2 ML PP ROUND BOTTOM INTERNALTHREAD greiner bio-oneCatalog #122263-2DG
Troubleshooting
Safety warnings
Please wear protective clothing, PPE and follow local safety guidelines.
Ethics statement
All research must adhere to the current, updated Helsinki declaration.
Prior ethical approval must be granted.
Informed consent must be given.
Before start
Ethics approvals, consent and local safety rules in place.
See section preparation below.

Preparation
Prepare Amount100 mL of ReagentDulbecco’s Phosphate Buffer Saline Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8537 +2%ReagentFetal Bovine Serum, Premium Gibco - Thermo Fisher ScientificCatalog #A5670701 for two blood vials (~Amount15 mL total) under the hood

Prepare Amount50 mL stock of Erylysis Buffer as follows: EDTAConcentration0.1 millimolar (mM) , NH4Cl Concentration155 millimolar (mM) , KHCO3 Concentration10 millimolar (mM) . Adjust the pH to 7.3 - 7.4 with HCl or NaOH. Erylysis buffer can be stored long term at Temperature4 °C

Bring ReagentLymphoprep™ 500 mL STEMCELL Technologies Inc.Catalog #7851 , ReagentDulbecco’s Phosphate Buffer Saline Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8537 +2%ReagentFetal Bovine Serum, Premium Gibco - Thermo Fisher ScientificCatalog #A5670701 solution, Erylysis buffer, ReagentCryoStor CS10-100mlSTEMCELL Technologies Inc.Catalog #07930 and Mr. Frosty at room temperature (Temperature18 °C - Temperature25 °C )

Add Amount15 mL ReagentLymphoprep™ 500 mL STEMCELL Technologies Inc.Catalog #7851 to the ReagentSepMate-50STEMCELL Technologies Inc.Catalog #85450 using a Amount25 mL pipette by carefully pipetting it through the central hole of the ReagentSepMate-50STEMCELL Technologies Inc.Catalog #85450 insert (plastic insert inside the tube).
Note
If needed, use a sterile (non-filter tip) Amount1000 µL pipette tip placed into the insert hole as a way to stabilize and guide the strip pipette and Lymphoprep solution through the hole.



PBMC isolation
Add Amount15 mL - Amount17 mL blood into a Reagent50ml Falcon tubesCorningCatalog #352070 . Add equal volume ofReagentDulbecco’s Phosphate Buffer Saline Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8537 +2%ReagentFetal Bovine Serum, Premium Gibco - Thermo Fisher ScientificCatalog #A5670701 to the Reagent50ml Falcon tubesCorningCatalog #352070 . Close the lid and mix gently by inverting 2 times.

Keeping the ReagentSepMate-50STEMCELL Technologies Inc.Catalog #85450 vertical, add the diluted blood sample by slowly pipetting it down the side of the tube. The sample will mix with the density gradient medium above the insert. Close the lid.

Centrifuge the ReagentSepMate-50STEMCELL Technologies Inc.Catalog #85450 at 1200xg for Duration00:10:00 at TemperatureRoom temperature with the brakes on (medium deceleration).

With care, remove the ReagentSepMate-50STEMCELL Technologies Inc.Catalog #85450 lid. To reduce platelet contamination, pipette off and discard about Amount10 mL of supernatant from the surface using a Amount10 mL pipette – well above the enriched mononuclear cell layer.

Pour the remaining top layer containing the enriched mononuclear cells into a new Reagent50ml Falcon tubesCorningCatalog #352070 by inverting the ReagentSepMate-50STEMCELL Technologies Inc.Catalog #85450 to 180 degrees. To avoid pouring the red blood cells (RBCs), do not hold the ReagentSepMate-50STEMCELL Technologies Inc.Catalog #85450 in the inverted position for longer than Duration00:00:02

Top up the enriched mononuclear cells in tube with ReagentDulbecco’s Phosphate Buffer Saline Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8537 +2% ReagentFetal Bovine Serum, Premium Gibco - Thermo Fisher ScientificCatalog #A5670701 to Amount50 mL and centrifuge at 300xg for Duration00:05:00 to pellet PBMCs

Carefully discard the supernatant using a vacuum aspirator without disturbing the PBMC pellet
To get rid of residual red blood cells (RBCs), resuspend the pellet in Amount5 mL -Amount7 mL of Erylysis Buffer and incubate at TemperatureRoom temperature for Duration00:05:00

Top up the remaining enriched mononuclear cells with ReagentDulbecco’s Phosphate Buffer Saline Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8537 +2% ReagentFetal Bovine Serum, Premium Gibco - Thermo Fisher ScientificCatalog #A5670701 in Reagent50ml Falcon tubesCorningCatalog #352070 and centrifuge at 300xg for Duration00:05:00 to pellet PBMCs

Carefully discard the supernatant using a vacuum aspirator without disturbing the PBMC pellet
Resuspend the PBMC pellet in Amount3 mL -Amount4 mL of ReagentCryoStor CS10-100mlSTEMCELL Technologies Inc.Catalog #07930 or ReagentFetal Bovine Serum, Premium Gibco - Thermo Fisher ScientificCatalog #A5670701 +10%ReagentDMSOPanreac AppliChemCatalog #A3672

Count the cell suspension manually or using an automated cell counter . Adjust the volume of freezing media to reach the desired concentration of PBMCs (1 - 20*10^6 cells/mL)

Prepare Amount0.5 mL -Amount1 mL aliquots in ReagentCRYO.S 2 ML PP ROUND BOTTOM INTERNALTHREAD greiner bio-oneCatalog #122263-2DG and place them in Mr. Frosty. Cryopreserved PBMCs should be transferred into liquid nitrogen for long term storage after at least 24 hours at Temperature-80 °C . If cells are being used for experimentation within a few weeks of harvesting, they can be kept at Temperature-80 °C .

Documentation
Document the following information:
ID number and date
Time the blood was received. Time at first centrifugation step. Time the PBMCs were frozen. Time/date of transfer to liquid nitrogen.
Cell number, concentration and number of cryovials.


Acknowledgements
Claudia Schulte, Christian Deuschle and Ann-Kathrin Hauser at The Hertie Institute for Clinical Brain Research and The Neurobiobank of The Hertie Institute for Clinical Brain Research for their constant support and teamwork across projects.

The Michael J Fox Foundation
The Michael J Fox Foundation PINK1/PRKN consortium