Sep 23, 2019
Isolation of peripheral blood mononuclear cells
- 1Universitas Airlangga
Protocol Citation: Devi Oktafiani 2019. Isolation of peripheral blood mononuclear cells. protocols.io https://dx.doi.org/10.17504/protocols.io.7j5hkq6
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: September 23, 2019
Last Modified: September 23, 2019
Protocol Integer ID: 27997
Abstract
Peripheral blood mononuclear cells (PBMCs) are chiefly lymphocytes and monocytes. PBMCs are separated from the whole blood by a density gradient centrifugation method using Ficoll-Paque.
Guidelines
- Use freshly collected EDTA blood. If plasma needs to be used for any other purpose, then remove the plasma and add equal volume
- The ratio between Ficoll Histopaque and blood should be 1:1 for human blood.
Materials
MATERIALS
1XPBS solution
Histopaque® or Ficoll-Paque
Before start
Ficoll Histopaque is stored at 4 °C. Before use the tube needs to be kept at room temperature for 1-2 h since PBMCs will get cold shock or sometimes aggregate if layered in pre-chilled Ficoll Histopaqueon blood sample.
Freshly blood collected in EDTA tube
Ficoll Histopaque (Sigma- Aldrich, catalog number: 10771; Sterile PBS
Collect 4 ml of human venous blood sample in EDTA tube and mix well by gently inverting the tube several times.
Take 5 ml of Ficoll Histopaque in a 15 ml centrifuge tube.
Gently layer the blood on the top of Ficoll Histopaque using a 1 ml auto pipette. The layering should be done very slowly that blood and Ficoll Histopaque should stay as two different layers.
Centrifuge the tubes (without any delay) for 30 min at 100 x g in 4 °C in a swing-out bucket. Fixed angle rotors also can be used but would require more caution when separating cells in interphase.
Aspirate the whitish buffy coat (about 1 ml) (PBMCs) formed in the interphase between histopaque and medium.
The cells in interphase need to be aspirated without delay. If the tubes are kept standing for more than 10 min, PBMCs from the interphase will get disturbed and start settling down.
Wash (centrifuge in 100 x g for 10 min) twice with 10 ml of sterile PBS
Remove PBS
Add PBS 1 mL and transfer (PBS and cells) into 1,5 mL new tube