Materials and Reagents

100 ml peripheral blood obtained via subcutaneous venepuncture
Heparin - to prevent clotting of blood
Histopaque-1077 (Sigma-Aldrich, catalog number: 10771 ) or any equivalent ficoll solution (density 1.077 g/ml)
Fetal calf serum (FCS)
PBS (chilled)
DC isolation kit (Miltenyi Biotec, catalog number: 130-094-487 )
Antibodies (BD biosciences unless stated otherwise)
1. Anti-TCRa/b-FITC (catalog number: 347773 ) - isotype with mouse IgG₁-FITC
2. Anti-CD19-FITC (catalog number: 553785 ) - isotype with IgG_2a-FITC
3. Anti-CD14-FITC (catalog number: 561712 ) - isotype with IgG_2a-FITC
4. Anti-HLA-DR-PE (catalog number: 556644 ) - isotype with IgG_2a-PE
MACS buffer (see Recipes)

Equipment

Magnetic-activated cell sorting (MACS) magnet
LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
Centrifuge (50 ml tube capacity)
50 ml polypropylene conical tubes (Falcon^TM)
14 ml snap-cap polypropylene tubes

Procedure

Mononuclear cell isolation
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 Falcon 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. May need to dilute (1:100). A sample is taken as pre-sort control for FACs analysis to determine enrichment.
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.
13. Incubate for 10 min at 4 °C.
14. Transfer to a 50 ml Falcon 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.
FACS staining
1. Using the cell count above. 10⁵ cells are pelleted (400 x g for 5 min) in FACs tubes and re-suspended in residual PBS in tube (approximately 50 µl).
2. Add 1 µl of normal mouse serum to cells and incubate for 10 min (RT).
3. Proceed directly to adding directly conjugated antibody to cells (or isotype matched controls) as dictated by antibody datasheet. Stain in presence of normal serum. Stain for 20 min at RT in the dark (unless IgM which is then done at + 4 °C). Have one tube unstained for setting the parameters for collection.
4. Wash cells in 10x PBS and then spin (400 x g; 5 min, RT). Resuspend in 500 µl and proceed to FACs analysis.
5. Cells are characterized by the expression of a panel of cell surface markers consistent with a DC phenotype. The cells should be HLA-DR^high whilst TCR, CD14 and CD19 negative. Cells will also express CD1a, CD86 and CD83^low.

Representative data

Figure 1. Isolation of PBMC on histopaque 1077

Figure 2. FACs analysis of putative DCs post isolation shown to be HLA-DR positive and CD14, 19 and TCRα/β negative

Recipes

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

References

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.