Isolation of Dendritic Cells from Human Peripheral Blood

Materials and Reagents

1.30 ml peripheral blood obtained via subcutaneous venepuncture
2.Heparin – to prevent clotting of blood
3.Histopaque-1077 (Sigma-Aldrich, catalog number: 10771 ) or any equivalent
ficoll solution (density 1.077 g/ml)
4.Fetal calf serum (FCS)
5.PBS (chilled)
6.DC isolation kit (Miltenyi Biotec, catalog number: 130-094-487 )
7.MACS buffer

Recipes

1. MACS buffer

PBS supplemented with 0.5% fetal calf serum and 2 mM EDTA

Equipment

1.Magnetic-activated cell sorting (MACS) magnet
2.LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
3.Centrifuge (50 ml tube capacity)
4.50 ml polypropylene conical tubes (Ucallm, catalog number: M2050)
5.14 ml flow tubes (Ucallm, catalog number: C7014)
6.Disposable pasteur pipettes (Ucallm, catalog number: J8213)
7.1000µL Tips (Ucallm, catalog number: L1110)

Procedure

1.Cool MACS magnet adaptor and LS column at 4 °C. LS columns have a total loading capacity of 2 x 10⁹ unpurified cells (which would allow the isolation of 10⁸ labelled cells).
2.Cool MACS Buffer and PBS on ice.
3.Dilute the blood 1:1 in PBS.
4.Add 15 ml histopaque-1077 (density 1.077 g/ml) to a 50 ml conical tube.
5.Carefully layer 30-35 ml of blood/PBS on top.
6.Spin at 800 x g for 20 min at RT. No brake on centrifuge.
7.Take WBC at interphase. To do this use a pasteur pipette. Squeeze the teat prior to adding to conical tube and then suck up the white fluffy looking layer resting on histopaque. Avoid aspirating excessive histopaque-1077 as it can prevent pelleting of cells.
8.Pellet cells at 300 x g for 10 min (RT). If there is a problem pelleting cells possibly due to contaminating lymphoprep then dilute a further 1:4 in PBS and repeat centrifugation step.
9.Wash in cold PBS 300 x g for 10 min. Add 50 ml of PBS to pelleted cells.
10.Count cells.
11.Centrifuge at 300 x g for 10 min (RT).
12.Re-suspend pellet in 300 µl of MACS buffer per 10⁸ total cells. So for 3 x 10⁹ the cells would be re-suspended in 9 ml. Add 100 µl of FcR Blocking reagent (in kit) per 10⁸ and 100 µl of Microbead dendritic antibody cocktail to flow tube.
13.Incubate for 10 min at 4℃.
14.Transfer to a 50 ml conical tube and wash in PBS. Spin 300 x g for 10 min (RT).
15.As spin ends, charge the column.
16.To charge the column insert LS column into magnet. Then run 3 ml of MACS buffer through the column. Discard flow through.
17.Re-suspend the centrifuged cells in 500 µl of MACS buffer per 10⁸ cells and add to column.
18.Collect flow through. Re-apply this flow through to the column. Collect again and then wash the column a further two times with 3 ml washes. The flow through represents the dendritic cell population.
19.Count cells.
20.Cells are then used for downstream application.

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. 1. Reeves, M. and Sinclair, J. (2014). Isolation and Characterisation of Dendritic Cells from Peripheral Blood. Bio-protocol 4(22): e1300. DOI: 10.21769/BioProtoc.1300.

1. 2. Reeves, M. B. and Sinclair, J. H. (2013). Circulating dendritic cells isolated from healthy seropositive donors are sites of human cytomegalovirus reactivation in vivo. J Virol 87(19): 10660-10667.

Manufacturer’s Link

Sigma-Aldrich: https://www.sigmaaldrich.com

Miltenyi Biotec: https://www.miltenyibiotec.com

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