Apr 17, 2026

Human Whole Blood PBMC Isolation

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3pez1l25/v1
Human Whole Blood PBMC Isolation
  • 1Yale University
Nicholas C Buitrago-Pocasangre
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3pez1l25/v1
Protocol Citation: Nicholas Buitrago-Pocasangre 2026. Human Whole Blood PBMC Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3pez1l25/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 09, 2021
Last Modified: April 29, 2026
Protocol  Integer ID: 55792
Keywords: PBMC isolation, Human Blood PBMC Isolation, PBMC, ASAPCRN, human whole blood pbmc isolation this protocol detail, human whole blood pbmc isolation, pbmc isolation, protocol detail, protocol
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000529
Abstract
This protocol details ASAP PBMC isolation.
Attachments
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Guidelines
Make sure your protocol includes use of human blood samples (Yale BSL2).
Protocol materials
DPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144
LymphoprepSTEMCELL Technologies Inc.Catalog #18060
Bambanker Cell Freezing MediaBulldog BioCatalog #BB05
K2EDTA Vacutainer TubesBecton Dickinson (BD)Catalog #366643
Safety warnings
Work under BSL2 conditions (yale)
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Procedure
1h 13m 3s
Collect ten blood in K2EDTA Vacutainer TubesBecton Dickinson (BD)Catalog #366643 from human

Centrifuge at 500 x g for 00:10:00 to separate plasma from whole blood.

10m
Procedure
1h 13m 3s
Remove 5 mL of plasma, mix by inversion or pipet and aliquot into 1 mL of plasma
Procedure
1h 13m 3s
Replace volume of removed plasma with equal volume ofDPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 .

Procedure
1h 13m 3s
Pour or pipet blood into each 50 mL conical tube. Each tube should be relatively equal volume.

Note
  • Do not exceed 20 mL of whole blood per 50 mL conical tube.
  • To maximize yield, using a 10 mL serological pipette, rinse blood tubes with 5 mL DPBS/2 tubes. Add PBS/blood mixture to collection 50 mL conical tube after rinses.




Procedure
1h 13m 3s
Make a 1:1 DPBS/Blood mixture.
Note
Fill up 50 mL tube with PBS to whatever twice the initial volume of whole blood was per tube.

Gently invert 50 mL tubes to mix.

Using a 10 mL serological pipet, aspirate 12.5 mL LymphoprepSTEMCELL Technologies Inc.Catalog #18060
Note
Gently place pipet into bottom of 50 mL conical and remove the entire pipette from pipet aid.


Wait for the pipette to drain.
Note
A layer of Ficoll will form at the bottom.

Lift pipette up and stop at the interface, wait 00:00:03 (for the last 1 mL of Ficoll to release), cap the pipette with your fingers, and continue to lift the pipette entirely out of the 50 mL conical tub.

3s
Uncap your finger from the pipette and transfer the liquid into the liquid waste.
Centrifuge 800 x g for00:20:00 acceleration at 4, deceleration at 4 Room temperature .

20m
After 00:30:00 , transfer PBMC layer to new 50 mL collection tube.

Note
Do not touch the red blood cell pellet.


30m
Remove as much of PBMC layer as you can on first pass, then move on to the next tube. After you have done all the tubes, repeat again as a second pass.


Note
If you position the pipette so that the end of the pipette is touching the 50 mL conical tube wall and the tip is touching the front of the tube, you can stabilize your hand this way.


Make a 1:1 PBMC layer/DPBS. Fill up 50 mL conical tube to whatever twice the volume of the PBMC layer, cap, and invert to mix.

Centrifuge 400 x g 00:08:00 max acceleration: 9 and max deceleration: 9 Room temperature .

8m
Pour off supernatant with one quick motion. Resuspend pellet with 10 mL PBS. If you have multiple tubes use the same 10 mL to resuspend and wash all 4. Repeat rinse with another 10 mL DPBS.
Fill up resuspended pellet up to50 mL DPBS.
Invert 50 mL conical tube to mix. If you are counting, aliquot 10 µL of resuspension.

Centrifuge 400 x g for 00:05:00 max acceleration: 9 and max deceleration: 9 Room temperature .
Note
During the spin, count cells and/or roughly estimate the total # of PBMCs you have. 1-2 million PBMCs/mL (whole blood) is the expected range.

5m
Pour off supernatant. Resuspend pellet 10-20e6 cells/ 1 mL Bambanker Cell Freezing MediaBulldog BioCatalog #BB05 and mix.

Transfer aliquots quickly to Room temperature Mr. Frosty with IPA and transfer to -80 °C .