Isolation of PBMC from Human Peripheral Blood

Materials and Reagents

1.Freshly collected heparinised blood
2.Ficoll Histopaque (Sigma-Aldrich, catalog number: 10771)
3.Sterile Dulbecco’s modified eagle medium (ILEX LIFE SCIENCES, catalog number: P04-03590)
4.Pencillin-streptomycin solution (Sigma-Aldrich, catalog number: P-4333)
5.Heparin vials (BD Biosciences, catalog number: 367886)

Recipes

1.Media
Dulbecco’s modified eagle medium supplemented with 1% of Pencillin-streptomycin solution

Equipment

1.Centrifuge machine with swing-out bucket rotor (Eppendorf 5810 R)
2.Sterile 15 ml centrifuge tubes (Ucallm, catalog number: M2015)
3.Auto pipettes and 1000µL tips (Ucallm, catalog number: L1010)

Procedure

1.Collect 4 ml of human venous blood sample in heparinised vials (BD biosciences) and mix well by gently inverting the tube several times.
2.Take 4 ml of Ficoll Histopaque in a 15 ml centrifuge tube.
3.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.
4.Centrifuge the tubes (without any delay) for 30 min at 100 x g at RT in a swing-out bucket. No brake on centrifuge. Fixed angle rotors also can be used but would require more caution when separating cells in interphase.
5.Aspirate the whitish buffy coat (about 1 ml) (PBMCs) formed in the interphase between histopaque and medium in a new 15 ml centrifuge tube.
6.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 media. The approximate yield of cells from 4 ml of blood varies between 10⁷-10⁸.

Technical notes

1.Use freshly collected heparinised blood. If plasma needs to be used for any other purpose, then remove the plasma and add equal volume of sterile Dulbecco’s modified eagle medium before proceeding further.
2.The ratio between Ficoll Histopaque and blood should be 1:1 for human blood.
The ratio could be different for purification of PBMC of other species.
3.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.

References

1.Panda, S. K. and Ravindran, B. (2013). Isolation of Human PBMCs. Bio-protocol 3(3): e323. DOI: 10.21769/BioProtoc.323.
2.Panda, S. K., S. Kumar, N. C. Tupperwar, T. Vaidya, A. George, S. Rath, V. Bal and B. Ravindran(2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.

Manufacturer’s Link

Sigma-Aldrich : https://www.sigmaaldrich.com
ILEX LIFE SCIENCES: https://ilexlife.com
BD Biosciences:https://www.bdbiosciences.com
Eppendorf: https://www.eppendorf.com

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