|Plasmacytoid DCs (pDC)||0.5 % spleen; 0.2 % lymph nodes||CD45, CD11c, MHC class II, mPDCA-1 (CD317 or BST2), Siglec-H, CD45R (B220), Ly-6C||Upon pathogen encounter, produce large amounts of type I IFN and acquire antigen-presenting capacity|
|Conventional DC 1 (cDC1)||0.5 % spleen; 0.1 % lymph nodes||CD45, CD11c, MHC class II, CD8a, XCR1, CD24, CLEC9A||Performs cross-presentation of antigens to MHC class I and start type I cytotoxic immune responses|
|Conventional DC 2 (cDC2)||1 % spleen; 0.2 % lymph nodes||CD45, CD11c, MHC class II, CD4, SIRPα, CD11b|
Many markers and functions of myeloid cells, such as expression of MHC class II, CD11c, CD11b, and antigen presentation, are shared among different myeloid cell types such that the classification and identification of myeloid cell remains an evolving subject in the scientific community. Moreover, cell subsets and markers are not always consistent between mice and humans leading to debate over the correspondence of mouse and human subsets.
The latest classification defines 3 mouse DC subsets in lymphoid organs, which have counterparts in human blood: plasmacytoid DC (pDC) and the 2 conventional or myeloid DCs (cDC) subsets cDC1s and cDC2s.
Plasmacytoid DCs (pDC) are primary 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 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).
Conventional DC 1 (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 Batf3 transcriptional factor and are characterized by different sets of markers depending on their location, origin, and function. Resident spleen and lymph node cDC1s express CD8a, XCR1, and CD24. When cDC1s migrate from non-lymphoid tissue to a draining lymph node, they are distinguished from resident cDCs by markers expressed in the periphery, such as CD103, by activation molecules, and 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 migratory and resident cDCs.
cDC1s recognize intracellular pathogens and start type 1 immune responses including ILC1, NK cells and T helper cell 1 skewing. Moreover, cDC1s efficiently cross-present extracellular antigens to CD8+ T cell and secrete IL-12, making them important for cytotoxic response to viral infections and tumors (PMID: 23516985).
Conventional DC 2 (cDC2) exhibit the same MHC class II and CD11c expression pattern as cDC1s, but express markers not present on cDC1s and depend on a different transcription factor, IRF4. Resident spleen and lymph node cDC2s express CD4 and SIRPα. Migratory cDC2s infiltrating 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).cDC2s induce different responses, such as activation of ILC2s and TH2 cells against parasites and during asthma, and induction of ILC3s and TH17 immune responses to extracellular bacteria (PMID: 27760337).
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. Enzymatic and mechanical dissociation of spleen is necessary for 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 gentleMACS™ Octo Dissociator with Heaters enables efficient, automated, and hands-free enzymatic and mechanical digestion of several spleens in parallel. Both cell recovery and epitope preservation are high. For more details, see corresponding spleen chapter.
Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of DCs cells and distinct DC subsets.For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic Cell Separation .
|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|
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. The 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.
Fast isolation of cross-presenting DCs from mouse spleen. XCR1+ DCs were isolated from a spleen single-cell suspension 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 and propidium iodide fluorescence. The dot plots on the right show conventional DCs gated on CD11c+MHC class II+ cells, stained for cross-presenting DC markers (CD8a and XCR1).
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.
|MHC class IIhigh||MHC class II+||MHC class II+||MHC class IIint|
|CD172a (SIRPα)+/–||CD370 (CLEC9A)||CD4+||Siglec-H+|
|CD205 (DEC205) +|
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 via specific receptors has been used to induce effective antigen-specific cell (APC) responses and thus, 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 like 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 (TLR) 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. TLR 7 and 8 are involved in responses to viral infection and recognize single-stranded RNAs as their natural ligand, as well as small synthetic molecules. TLR 7 is expressed on murine myeloid dendritic cells and monocytes, while TLR 8 is functionally impaired in mice.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.
|Cell subset||Frequency (percent of total CD45+ cells)||Markers||Function|
|Plasmacytoid DCs (pDC)||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, produce large amounts of type I IFN and acquire antigen-presenting capacity|
|Conventional DC 1 (cDC1)||1 % skin, 0.5 % lung, 1 % intestine||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 % skin, 1.5 % lung, 0.45 % intestine||CD45, CD11c, MHC class II, SIRPα, CD11b|
|Inflammatory DCs||Variable, it depends on inflammation||CD11c, MHC class IIMo-DCs: CD64, F4/80, MER(MERTK), CD11b||Induce naïve T cell activation|
|Langerhans cells||30–50 % skin||Located in skin to take up microbial antigens and become APCs|
The complexity of DC phenotyping makes it essential that all epitopes needed to identify subsets are preserved during tissue digestion. Miltenyi Biotec offers optimized kits for the dissociation of tissues and preparation of single-cell suspensions from several tissues, including lamina propria, lung, and skin in combination with the gentleMACS™ Octo Dissociator with Heaters.
|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|
The CD11c MicroBeads UltraPure, mouse are optimized for the rapid and simple 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 with 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.
For a comprehensive analysis of DCs in non-lymphoid tissues, expand 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) with markers such as CD64 and F4/80 that should be negative in lymphoid counterpart subsets.
|Lymphoid tissue cDC||Nonlymphoid tissue cDC|
|pDC||CD8+ cDC||CD11b+ cDC||CD103+ CD11b+ cDC||CD103+ CD11b+ intestinal cDC||CD103+ CD11b+ cDC|
|MHC class II||+||++||++||++||++||++||++|
|CD64 (Fc r1)||-||-||-||-||-||++||ND|