Materials and Reagents

Freshly collected heparinised blood
Ficoll Histopaque (Sigma- Aldrich, catalog number: 10771 ; MP Biomedicals, catalog number: 091692254 )
Sterile PBS or Dulbecco's modified eagle medium (catalog number: P-04-03590)
Pencillin-streptomycin solution (Sigma- Aldrich, catalog number: P-4333 )
Heparin vials (BD Biosciences, catalog number: 367886 )
Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution (see Recipes)

Equipment

Centrifuge machine with swing-out bucket rotor (Eppendorf 5810 R )
Sterile 15 ml centrifuge tube
Auto pipettes and 1 ml tips

Procedure

Collect 4 ml of human venous blood sample in heparinised vials (BD biosciences) and mix well by gently inverting the tube several times.
Take 4 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 or sterile Dulbecco's modified eagle medium. The approximate yield of cells from 4 ml of blood varies between 10⁷-10⁸.

Recipes

Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution

Technical notes

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.
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.
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.

Acknowledgments

The laboratory protocol was evolved over time in the senior authors’ laboratory using a template that was published in 1986 in Handbook of Experimental Immunology / edited by D. M. Weir; co-editors, L. A. Herzenberg, Caroline Blackwell, Leonore A. Herzenberg. Blackwell Scientific Publications. Institute of Life Sciences is funded by Department of Biotechnology, Govt of India and SP was supported by a fellowship grant from Indian Council of Medical Research.

References

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.