Anti-Mouse Dendritic Cells – Purified in vivo PLATINUM™ Functional Grade

Clone 33D1 is a monoclonal antibody widely used in in vivo mouse studies to specifically target and deplete a major subset of mouse dendritic cells (DCs), especially the CD8?, CD4+ conventional dendritic cell type 2 (cDC2) population.

Essential context and supporting details:

• Mechanism of Use: 33D1 binds specifically to DCIR2 (also known as Clec4a4), a surface antigen highly expressed on mouse cDC2s. When administered with rabbit complement, 33D1 antibody induces cell lysis, allowing for selective depletion of these dendritic cells in living mice or tissue preparations.

• Typical Applications:

  • Selective DC Depletion: By depleting DCIR2+ (33D1+) dendritic cells, researchers study their role in immune responses, for example, in primary T cell stimulation or in disease models like autoimmunity, obesity, and infections.
  • Functional Immune Assays: Removal of DCs via 33D1 and complement has been shown to reduce the stimulatory capacity of spleen suspensions in mixed leukocyte reactions and to attenuate both proliferative and cytotoxic T cell responses, confirming the critical role of these cells in immune priming.
  • In Vivo Modulation: Recent studies also use 33D1 antibody to delineate the contribution of cDC2s in the progression or amelioration of diseases with strong inflammatory or autoimmune components. For instance, depleting DCIR2+ cells can alter the course of experimental models such as experimental autoimmune encephalomyelitis (EAE) and diabetes.

• Specificity: 33D1 reacts with dendritic cells in mouse thymus, spleen, lymph nodes, Peyer’s patches, and can be used to monitor or purify these cells from complex lymphoid mixtures. The antibody does not react with macrophages, granulocytes, lymphocytes, or other major leukocyte populations.

• Form and Purity: For in vivo use, 33D1 is typically provided as a purified rat IgG2b antibody, processed to be of low endotoxin content and suitable for functional antibody studies in mice.

• Other Uses: In addition to depletion, fluorescent or biotin-labeled 33D1 can be used for flow cytometry or immunohistochemistry to identify and sort dendritic cell subsets in mouse tissues.

In summary, clone 33D1 is primarily used in vivo to selectively deplete or track mouse DCIR2+ (cDC2) dendritic cells to investigate their immune functions, using direct antibody application often in combination with complement for cell ablation.

The correct storage temperature for sterile packaged clone 33D1 (anti-mouse dendritic cell marker antibody) depends on the intended storage duration:

• For long-term storage (up to 12 months), store at -20°C to -70°C in a manual defrost freezer and avoid repeated freeze-thaw cycles.
• For short-term storage (up to 1 month), store at 2°C to 8°C as supplied.

Ensure you follow any additional instructions provided by the manufacturer regarding light protection or dilution. Immediate storage upon receipt at the recommended temperature is essential for maintaining product stability.

Commonly, when researchers use 33D1 (anti-DCIR2) to identify or deplete conventional type 2 dendritic cells (cDC2) in mice, they pair it with other antibodies and markers to distinguish among dendritic cell subsets or to interrogate immune cell phenotypes. The most frequently used additional antibodies or proteins in the literature include:

• CD11c: A pan-dendritic cell marker, critical for confirming dendritic cell identity and distinguishing DCs from other cell types.
• CD8?: Used to distinguish cDC1 (CD8?+) from cDC2 (33D1+ DCIR2+), since 33D1 labels cDC2, which are typically CD8?-.
• F4/80: A macrophage marker used in combination to exclude macrophages from dendritic cell analysis, as 33D1 does not stain macrophages.
• MHC class II (I-A/I-E): Used to confirm antigen-presenting cell (APC) status and for functional analyses involving dendritic cells.
• CD45: A general leukocyte marker, often used alongside other markers to gate total immune cells in flow cytometry.
• B220 (CD45R): Used to identify and exclude plasmacytoid dendritic cells (pDCs), which are B220+.

These combinations allow researchers to carefully define DC subpopulations, for example:

Experimental applications (e.g., flow cytometry, immunohistochemistry, in vivo depletion) often combine 33D1 with these markers to purify, quantify, or functionally characterize distinct mouse DC subtypes.

Summary of the most common antibodies/proteins used with 33D1 in the literature:

• CD11c (pan-DC marker)
• CD8? (cDC1 marker)
• F4/80 (macrophage exclusion)
• MHC class II (APC confirmation)
• B220 (pDC exclusion)
• CD45 (leukocyte gating)

These combinations provide clear distinction among DC subpopulations and exclude contaminating cell types, which is critical for high-specificity immunological studies.

Key Findings from Clone 33D1 Scientific Literature

Identification and Characterization of 33D1 Antigen

• The 33D1 monoclonal antibody recognizes a murine antigen that is now known as Dendritic Cell Inhibitory Receptor 2 (DCIR2) or C-type lectin domain family 4, member a4 (Clec4a4), a 236 amino acid protein with a predicted molecular weight of 27.3 kDa.
• The expression of the 33D1 antigen is primarily observed in conventional type 2 dendritic cells (cDC2) within mouse thymus, spleen, lymph nodes, and Peyer's patches.
• Expression of 33D1 antigen can be induced in bone marrow-derived dendritic cells by GM-CSF and downregulated by IL-4.
• Binding of the 33D1 antibody to its antigen is calcium-dependent, which has practical implications for experimental protocols (e.g., staining in EDTA-free buffers).

Biological Functions of DCIR2 (33D1 Antigen)

• DCIR2, recognized by clone 33D1, is involved in antigen recognition and modulates T cell responses, particularly by suppressing autoimmunity through down-regulation of T cell priming.
• DCIR2-expressing dendritic cells have been found to play a role in ameliorating diseases with strong immune-inflammatory components, such as experimental autoimmune encephalomyelitis (EAE), experimental melanoma, and diabetes.
• These cells may also be implicated in the progression of diet-induced obesity and inflammation.

Technical and Application Insights

• Clone 33D1 has been widely used both in vitro and in vivo for the identification, depletion, and functional study of DCIR2+ dendritic cell populations in mouse models.
• The antibody is compatible with flow cytometry, immunofluorescence, and immunohistochemical staining, with specific recommendations for optimal staining conditions (e.g., calcium-containing buffers for FACS).
• Most N418+ dendritic cells in the mouse spleen are also 33D1 positive, suggesting overlap in marker expression among certain dendritic cell subsets.

Summary Table: Key Findings

Caveats and Limitations

• The precise molecular structure of the 33D1 antigen was initially unknown and has only recently been identified as DCIR2.
• While 33D1 is a robust marker for murine cDC2, its expression and functional roles may vary with tissue and inflammatory context.

These findings underscore the importance of clone 33D1 as a tool for studying dendritic cell biology, particularly in the context of immune regulation, autoimmunity, and inflammation in mouse models.