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As sentinels of the immune system, dendritic cells (DCs) are naturally located in all tissues, especially those at the interface between the body and the external environment, such as skin, lung, and intestine. They are also present in lymphoid organs where they activate T cells, thus initiating immune responses. Due to limited access to human solid tissues, most of the studies on DCs from lymphoid and non-lymphoid organs have been performed on tissues obtained from mouse models.
At a glance: DC cell subsets from lymphoid tissue (spleen and lymph nodes)
| Cell subset | Frequency | Markers | Function |
| Plasmacytoid DCs (pDCs) | 0.5% in spleen; 0.2% in lymph nodes | CD45, CD11c, MHC class II, mPDCA-1 (CD317 or BST2), Siglec-H, CD45R (B220), Ly-6C | Upon pathogen encounter, pDCs produce large amounts of type I IFN and acquire antigen-presenting capacity. |
| Conventional DC 1 (cDC1) | 0.5% in spleen; 0.1% in lymph nodes | CD45, CD11c, MHC class II, CD8a, XCR1, CD24, CLEC9A | cDC1 perform cross-presentation of antigens to MHC class I and start type I cytotoxic immune responses. |
| Conventional DC 2 (cDC2) | 1% in spleen; 0.2% in lymph nodes | CD45, CD11c, MHC class II, CD4, SIRPα, CD11b | cDC2 activate innate lymphoid cells 2 (ILC2) and TH2 cells and Induce ILC3 and TH17 immune responses. |
Different myeloid cell types have many markers and functions in common, such as expression of MHC class II, CD11c, CD11b, and antigen presentation. Therefore, the classification and identification of myeloid cells remain evolving subjects in the scientific community. Moreover, cell subsets and markers are not always consistent between mouse and human, leading to debates over the correspondence of mouse and human subsets.
The latest classification defines three mouse DC subsets in lymphoid organs, which have counterparts in human blood, namely pDCs and the two conventional or myeloid DC (cDC) subsets cDC1 and cDC2.
pDCs are primarily located in blood and lymphoid tissues. They depend on the E2-2 transcription factor and express B220, Siglec-H, mPDCA-1 (CD317 or Bst2), as well as intermediate levels of MHC class II, CD11c, and costimulatory molecules. pDCs are poor stimulators of T helper (TH) cells, but upon stimulation with bacterial DNA containing particular unmethylated CpG motifs or upon viral challenge, they produce large amounts of type I IFN and acquire antigen-presenting capacity (PMID: 16172135, 15728491).
cDC1 are located in both lymphoid and non-lymphoid tissues, and express higher levels of MHC class II and CD11c compared to pDCs. They show dependence on the Batf3 transcription factor and are characterized by different sets of markers depending on their location, origin, and function. Resident spleen and lymph node cDC1 express CD8a, XCR1, and CD24. When cDC1 migrate from non-lymphoid tissue to a draining lymph node, they can be distinguished from resident cDC by markers expressed in the periphery such as CD103, by activation molecules, by higher levels of MHC class II, and lower levels of CD11c. However, when resident cDCs are activated during inflammation, these markers cannot be used to discriminate between migratory and resident cDCs.
cDC1 recognize intracellular pathogens and start type 1 immune responses including ILC1, NK cells, and T helper cell 1 induction. Moreover, cDC1 efficiently cross-present extracellular antigens to CD8+ T cells and secrete IL-12, making them important for cytotoxic responses to viral infections and tumors (PMID: 23516985).
cDC2 exhibit the same MHC class II and CD11c expression pattern as cDC1, but express additional markers not present on cDC1 and depend on a different transcription factor, i.e., IRF4. Resident spleen and lymph node cDC2 express CD4 and SIRPα. Migratory cDC2 that infiltrate lymph nodes can be distinguished from resident cDCs under non-inflammatory conditions by the expression of MHC class II, CD11c, and peripheral and migratory markers (e.g. CCR7 and maturation markers).
cDC2 induce different responses, such as activation of ILC2 and TH2 cells against parasites and during asthma, and induction of ILC3 and TH17 immune responses to extracellular bacteria (PMID: 27760337).
Miltenyi Biotec has created dedicated application protocols for working with mouse DCs.
Research on mouse DCs is hampered by difficulties in isolating viable cells from solid tissues in numbers that allow comprehensive downstream investigation, such as flow cytometry analysis, cell sorting, and transcriptional studies. One critical aspect is the reliable dissociation of tissues, yielding cells with high viability and epitope integrity. Both enzymatic and mechanical dissociation of spleen is necessary to achieve a high recovery of DCs which are trapped in connective tissues and are inadequately released by simple mechanical meshing (PMID: 23516985).
The Spleen Dissociation Kit, mouse in combination with the gentleMACS™ Octo Dissociator with Heaters enables efficient, automated, and hands-free enzymatic and mechanical dissociation of several spleens in parallel. Both cell recovery and the level of epitope preservation are high in the resulting single-cell suspensions. For more details, see the corresponding spleen chapter.
Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of DCs and distinct DC subsets.
For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation .
MACS Handbook:
Magnetic cell separation
At a glance: Kits and reagents for the isolation of various DC subsets
| Cell subset | Isolation strategy | Comments | Automation | Product |
| Pan DCs | Positive selection of target cells | Yes* | CD11c MicroBeads UltraPure, mouse | |
| Pan DCs | Depletion of non-target cells | Isolation of all untouched DC subpopulations from spleen | Yes* | Pan Dendritic Cell Isolation Kit, mouse |
| Pan DCs | Positive selection of target cells | Mixture of CD11c and Anti-mPDCA-1 MicroBeads | Yes* | Pan DC MicroBeads, mouse |
| CD4+ DCs | Depletion of non-DC cells followed by positive selection of target cells | Isolation of cDC2 subset from spleen | Yes* | CD4+ Dendritic Cell Isolation Kit, mouse |
| CD8+ DCs | Positive selection of target cells | Isolation of cDC1 subset from spleen | Yes* | Anti-XCR1 MicroBead Kit (Spleen), mouse |
| CD8+ DCs | Depletion of non-DC cells followed by positive selection of target cells | Isolation of cDC1 subset from spleen | Yes* | CD8+ Dendritic Cell Isolation Kit, mouse |
| pDCs | Positive selection of target cells | Isolation of pDCs in non-inflammed tissues | Yes* | Anti-mPDCA-1 MicroBeads, mouse |
| pDCs | Depletion of non-target cells | Isolation of untouched pDCs | Yes* | Plasmacytoid Dendritic Cell Isolation Kit, mouse |
| *Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. | ||||
DCs constitutively express the hematopoietic surface markers CD45, MHC class II, Flt3, and CD11c, and lack T cell, natural killer (NK) cell, B cell, granulocyte, and erythrocyte lineage markers (PMID: 23516985). In mice, CD11c is a well-established, yet not exclusive DC marker. Pan DCs can be enriched by positive selection from spleen using CD11c MicroBeads UltraPure, mouse or isolated by depletion of non-target cells using the Pan Dendritic Cell Isolation Kit, mouse. Subset populations can be further enriched by a second separation step using MicroBeads or flow cytometry–based cell sorting.
Isolation of total untouched DCs. Pan DCs were isolated from a mouse spleen cell suspension using the Pan Dendritic Cell Isolation Kit, mouse, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with CD11c-APC and mPDCA-1-FITC and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris, dead cells, and lineage-positive cells were excluded from the analysis based on scatter signals, propidium iodide fluorescence, and lineage antigen expression.
The cDC2 subset of DCs co-expresses CD11c, CD11b, MHC class II, CD40, CD80, and CD86 and is negative for CD8 and CD205. cDC2 represent the major population of DCs in spleen (56% of all CD11c+ cells) and constitute only a small population in peripheral lymph nodes (4% of all CD11c+ DCs). The CD4+ Dendritic Cell Isolation Kit, mouse isolates CD4+ cells from an enriched DC sample, after depletion of non-DC cells.
Two-step isolation of CD4+ DCs from a mouse spleen. CD4+ DCs were isolated from a spleen cell suspension using the CD4+ Dendritic Cell Isolation Kit, mouse, an LD and two MS Columns, a MidiMACS™ and a MiniMACS™ Separator. Cells were fluorescently stained with the antibodies as indicated and analyzed by flow cytometry. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
In the past, studies of cDC1 were hampered by the low frequency of this cell type and a lack of specific cell surface markers. Recently, however, the receptors Clec9A and XCR1 have been identified as specific markers for cross-presenting cDCs in lymphoid and non-lymphoid tissue. The Anti-XCR1 MicroBead Kit (Spleen), mouse represents a novel method for the fast and easy isolation of cross-presenting DCs, that allows routine enrichment of XCR1+ DCs with high recovery and purity, without pre-depletion of non-target cells or laborious cell sorting.
Fast isolation of cross-presenting DCs from mouse spleen. XCR1+ DCs were isolated from a single-cell suspension of dissociated spleen tissue using the Anti-XCR1 MicroBead Kit (Spleen) with two MS Columns and a MiniMACS™ Separator. Cells were fluorescently stained with Anti-MHC Class II-VioGreen™, CD11c-VioBlue®, Anti-XCR1-PE, and CD8a-APC and analyzed using the MACSQuant Analyzer 10. Cell debris, dead cells, and autofluorescent cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. The dot plots on the right show conventional DCs gated on CD11c+MHC class II+ cells, stained for markers of cross-presenting DCs (CD8a and XCR1).
pDCs can be isolated from spleen by positive selection using the Anti mPDCA-1 MicroBeads, mouse or by depletion of unwanted cells with the Plasmacytoid Dendritic Cell Isolation Kit, mouse, which was designed for the rapid enrichment of pDCs.
Depletion of non-target cells generates a highly enriched population of pDCs. pDCs were isolated from splenocytes using the Plasmacytoid Dendritic Cell Isolation Kit, a MidiMACS Separator, and an LS Column. Cells were fluorescently stained with Anti-mPDCA-1-FITC and Anti-Siglec-H-PE and analyzed by flow cytometry on the MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
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Phenotyping or enriching DCs based on surface markers can be challenging because several myeloid cells share similar marker patterns, and different DC subsets can strongly modify their surface marker expression depending on their location and activation status. Furthermore, enzymatic tissue dissociation can affect surface epitopes. Optimization of antibodies with a specific and consistent enzyme cocktail is important to avoid false negative staining that compromises the identification of specific DC subsets.
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At a glance: Markers for the detection of DCs from lymphoid tissue by flow cytometry
| Pan DCs | cDC1 | cDC2 | pDCs |
| CD11c+ | CD11c+ | CD11c+ | CD11cint |
| MHC class IIhigh | MHC class II+ | MHC class II+ | MHC class IIint |
| CD11b+/– | CD8a+ | CD8a+ | CD317 (BST2)+ |
| CD172a (SIRPα)+/– | CD370 (CLEC9A) | CD4+ | Siglec-H+ |
| CD4+/– | XCR1+ | CD11b+ | CD45R (B220)+ |
| CD8+/– | CD4– | CD172a (SIRPα)+ | CD11b– |
| XCR1+/– | CD11b– | Ly-6Chigh | |
| F4/80– | CD172a (SIRPα)– | ||
| CD205 (DEC205) + |
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DCs can be cultured in vitro and stimulated by antigen or toll-like receptor (TLR) ligand presentation.
At a glance: Kits and reagents for the stimulation of DCs from lymphoid tissue
| Use | Comments | Product |
| Stimulation | Antigen-specific activation of DCs; available in research, premium, and GMP Grades | PepTivator Peptide Pools |
| Stimulation | TLR ligand presentation | TLR7/8 Agonists |
Effective stimulation of antigen-specific T cells is achieved reliably with PepTivator® Peptide Pools. This extensive panel of virus- or tumor-specific antigens consists of 15-mer peptides with 11-amino-acid overlaps, covering the complete sequence of the respective antigen.
Antigen targeting of antigen-presenting cells (APCs) via specific receptors has been used to induce effective antigen-specific cell responses and characterize the function of new receptors on APCs and compare these with well-known receptors. The Ova Antigen Delivery Module Set was developed for in vitro targeting of ovalbumin to APCs such as DCs. The set includes all reagents needed for the isolation of DCs, for antigen delivery, and for subsequent analysis of antigen presentation.
Toll-like receptors (TLRs) recognize highly conserved structural motifs of a wide variety of ligands expressed exclusively by microbial pathogens, called pathogen-associated molecular patterns (PAMPs). This function is part of an early innate immune response. TLR7 and 8 are involved in responses to viral infection and recognize single-stranded RNAs as their natural ligand as well as small synthetic molecules.
The TLR7/8 Agonists ORN R-0006, ORN R-2336, ORN RNA 40, and ORN R-2176-dT activate murine TLR7 and induce secretion of a broad spectrum of cytokines, including IFN-α, IFN-γ, IL-12, and TNF-α.
MACS Handbook:
Cell culture
At a glance: Kits and reagents for the generation of BM-DCs
| Use | Comments | Product |
| DC generation | Can be combined with IL-4 | Mouse GM-CSF |
| DC generation | Mouse Flt3-Ligand |
Murine bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow progenitor cells by culturing a total cell suspension from mouse bone marrow with specific cytokines. For example, BM-DCs that resemble conventional DCs can be generated using granulocyte/macrophage colony-stimulating factor (GM-CSF) alone, or GM-CSF in conjunction with interleukin-4 (IL-4). Alternatively, pDCs can be generated using Flt3-Ligand. These culture methods generate large numbers of DCs, but also result in heterogeneous DC populations.
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MACS Cytokines and cell culture reagents (brochure)
Each DC subset resident in spleen and lymph nodes (pDCs, cDC1, and cDC2) has a corresponding subset in non-lymphoid tissues that shares the same ontogeny and presents a similar but not identical phenotype (PMID: 23516985). This peripheral phenotype, in addition to certain activation markers, often allows the distinction of counterpart cells migrating into non-inflamed lymphoid tissues.
MACS Handbook:
Sample preparation
At a glance: DC cell subsets from non-lymphoid tissues
| Cell subset | Frequency (percentages refer to total CD45+ cells) | Markers | Function |
| Plasmacytoid DCs (pDCs) | Rare at steady state, variable during inflammation | CD45, CD11c, MHC class II, mPDCA-1 (CD317 or BST2),Siglec-H, CD45R (B220), Ly-6C | Upon pathogen encounter pDCs produce large amounts of type I IFN and acquire antigen-presenting capacity |
| Conventional DC 1 (cDC1) | 1% in skin, 0.5% in lungs, 1% in intestines | CD45, CD11c, MHC class II, CD103, XCR1, CD24, CLEC9A | Perform cross-presentation of antigens to MHC class I and start type I cytotoxic immune responses |
| Conventional DC 2 (cDC2) | 25% in skin, 1.5% in lungs, 0.45% in intestines | CD45, CD11c, MHC class II, SIRPα, CD11b | Activate ILC2 and TH2 cells and induce ILC3 and TH17 immune responses |
| Inflammatory DCs | Variable, depending on inflammation | CD11c, MHC class II Mo-DCs: CD64, F4/80, MER (MERTK), CD11b | Induce activation of naive T cells |
| Langerhans cells | 30–50% in skin | Located in skin to take up microbial antigens and become APCs |
The complexity of DC phenotyping makes it essential that all epitopes that are needed to identify subsets are preserved during tissue dissociation. Miltenyi Biotec offers optimized kits for the preparation of single-cell suspensions from several tissues, including lamina propria, lung, and skin. These kits together with the gentleMACS™ Octo Dissociator with Heaters provide optimal conditions for a combined enzymatic and mechanical tissue dissociation.
MACS Handbook:
Sample preparation
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At a glance: Kits and reagents for the separation of DCs from non-lymphoid tissues
| Cell subset | Isolation strategy | Comments | Automation | Product |
| Pan DCs | Positive selection of target cells | Can be used for various tissues, including lung and lamina propria | Yes | CD11c MicroBeads UltraPure, mouse |
| Langerhans cells | Positive selection of target cells | Yes | Epidermal Langerhans Cell MicroBead Kit, mouse | |
| *Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. | ||||
The CD11c MicroBeads UltraPure, mouse are optimized for the rapid and easy isolation of mouse DCs from single-cell suspensions of lymphoid and non-lymphoid tissues, such as lung and lamina propria. These MicroBeads greatly improve recovery and purity of the sorted population by specifically enriching viable cells.
Langerhans cells are best enriched by the Epidermal Langerhans Cell MicroBead Kit, mouse.
Langerhans cells isolated from a mouse epidermis single-cell suspension. A single-cell suspension was prepared using the Epidermis Dissociation Kit, mouse. The Epidermal Langerhans Cell MicroBead Kit, one MS Column, and a MiniMACS Separator were used to isolate the Langerhans cells, which were fluorescently stained with CD207 and CD11c and analyzed by flow cytometry on the MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
MACS Handbook:
Magnetic cell separation
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For a comprehensive analysis of DCs in non-lymphoid tissues, the list of markers described for the characterization of DCs from lymphoid tissue (see 2.5. Characterization of DCs from lymphoid tissue by flow cytometry) can be expanded by including CD64 and F4/80. These markers should not be expressed in the lymphoid counterpart subsets.
At a glance: Markers for the identification of DCs from lymphoid and non-lymphoid tissues
| Marker | pDCs | Lymphoid tissue cDCs | Non-lymphoid tissue cDCs | Langerhans cells | |||
| CD8+ cDC | CD11b+ cDC | CD103+ CD11b+ cDC | CD103+ CD11b+ intestinal cDC | CD103+ CD11b+ cDC | |||
| CD45 | + | + | + | + | + | + | + |
| CD11c | + | +++ | +++ | ++ | ++ | ++ | ++ |
| MHC class II | + | ++ | ++ | ++ | ++ | ++ | ++ |
| CD8 | subset | + | – | – | – | – | – |
| CD4 | + | – | +/– | – | – | – | ND |
| CD11b | – | – | + | – | + | + | + |
| CD103 | – | subset | – | ++ | ++ | – | – |
| Langerin | – | subset | – | + | – | – | ++ |
| EpCAM | – | – | – | – | – | – | ++ |
| B220 | + | – | – | – | – | – | - |
| CD24 | ND | ++ | + | ++ | ++ | +/– | ++ |
| Btla | + | ++ | + | ++ | + | +/– | ND |
| c-kit | – | + | + | + | + | +/– | ND |
| CD26 | + | + | + | + | + | +/– | ND |
| Xer1 | – | + | – | + | – | – | – |
| CD36 | – | + | – | + | ND | – | – |
| Cystatin C | + | ++ | + | ND | ND | ND | ND |
| Clec9a (DNGR1) | + | ++ | – | ++ | – | – | – |
| Cadm1 (Necl2) | – | + | – | ND | ND | ND | ND |
| CD205 | – | ++ | + | ++ | ND | ND | ++ |
| CX3CR1 | – | subset | – | – | – | ++ | + |
| CD209 (dc-sign) | ++ | – | + | – | + | +/– | – |
| F4/80 | – | – | + | – | – | + | + |
| CD172a (Sirpa) | + | – | ++ | – | – | ++ | + |
| CD64 (Fc r1) | – | – | – | – | – | ++ | ND |
| Ly-6C | ++ | – | – | – | +/– | – | |
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