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General Protocols

Primary DCs can be isolated from blood, and lymphoid or peripheral tissues, but the presence of DCs in blood is scarce and they are difficult to obtain in sufficient numbers from tissues. In addition, one may encounter large donor variations. The use of cell lines would overcome these challenges, but may hold insufficient similarity with primary DCs with respect to their response to environmental stimuli, ability to capture antigens, mature, migrate, produce cytokines, present antigens and activate T cells. Here we summarize a series of commonly used functional DC assays below and discuss the performance of cell lines and primary cells (summarized in Table 17.2).

DC Cell Lines

Most DC cell lines were differentiated from leukemia-derived cells, originating from myelogenous or monocytic lineage: the cytokine independent cell lines THP-1, U937, KG-1, HL-60 and the cytokine dependent cell line Mutz-3 are the most commonly used (Table 17.1) (Berges et al. 2005; van Helden et al. 2008; Santegoets et al. 2008). The use of these lines has the advantage of the availability of large numbers of synchronized cells with a long life span, but the association with selective DC subsets in peripheral blood and mucosal surfaces is largely unclear. All of these cells are leukemic, representing DC precursor stages and may often need the addition of specific growth and differentiation factors to induce at least some characteristics of differentiated DCs. MUTZ-3 is suggested to have the highest DC differentiation capacity (Santegoets et al. 2008) and may therefore be most representative for in vivo DCs.

Table 17.1 DC cell lines

Cell line

Derived from

DC

differentiation

T cell activation capacity

References

THP-1

Monoblastic leukemia cell line

Low

Yes

Tsuchiya et al. (1980) Chap. 14

U937

Monocytic leukemia cells

Moderate

Yes

Chapter 15

KG-1

CD34 myelomonocytic cell line

Incomplete

Yes

Koeffler et al. (1981)

HL-60

Acute pro-myelocytic leukemia cell line

Moderate

Yes

Koski et al. (1999)

MUTZ-3

Myeloid leukemia cell line

Relatively high

?

Hu et al. (1996)

Isolating Primary DCs from Blood

DCs can be directly isolated from PB using their unique surface markers (Fig. 17.1) and utilizing either magnetic bead separation or flow cytometric cell sorting (Vremec and Shortman 1997; Dzionek et al. 2000; Shortman and Liu 2002). Since the percentage of DCs in PB is generally low (cDC: 0.6 % and pDC 0.2 % of all PBMC), DC-enrichment (by depletion of cells expressing lineage markers CD3, CD14, CD16, CD19, CD20, and CD56) is often used. For magnetic bead sorting, separation/isolation kits are commercially available from companies such as Miltenyi Biotec and StemCell Technologies. Preferably, freshly isolated PBMCs obtained by a density gradient of Ficoll-Hypaque, are suspended in cell isolation buffer as per manufacturer's instructions. To optimize survival of primary DCs after isolation it may be helpful to add survival factors to the culture medium: 800 U/ml GM-CSF for cDCs, like CD1c or CD141 DCs, or 10 ng/ml IL-3 for pDCs.

Culturing peripheral blood-derived CD1c (BDCA1) DCs for 2 days with GM-CSF, vitamin D and Retinoic Acid (RA) induced high expression of RALDH as well as the ability to induce gut-homing receptors resembling the gut-resident CD103+Sirpα+ (DP) DCs to some extent (Sato et al. 2013).

Although using primary DCs directly isolated from the blood is feasible for most academic research labs and will probably resemble the in vivo DC biology most closely, the limited amount of primary DCs in PB is an obvious obstacle when many conditions and functional effects need to be assessed.

 
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