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

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

Product No.: D212

[product_table name="All Top" skus="D112"]

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Clone
33D1
Target
Dendritic Cells
Formats AvailableView All
Product Type
Monoclonal Antibody
Alternate Names
DC Marker, 33D1, DCIR2 (dendritic cell inhibitory receptor 2)
Isotype
Rat IgG2b κ
Applications
in vivo
,
WB

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Antibody Details

Product Details

Reactive Species
Mouse
Host Species
Rat
Recommended Isotype Controls
Recommended Dilution Buffer
Immunogen
Dendritic cells
Product Concentration
≥ 5.0 mg/ml
Endotoxin Level
<0.5 EU/mg as determined by the LAL method
Purity
≥98% monomer by analytical SEC
>95% by SDS Page
Formulation
This monoclonal antibody is aseptically packaged and formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.2 - 7.4 with no carrier protein, potassium, calcium or preservatives added. Due to inherent biochemical properties of antibodies, certain products may be prone to precipitation over time. Precipitation may be removed by aseptic centrifugation and/or filtration.
Product Preparation
Functional grade preclinical antibodies are manufactured in an animal free facility using in vitro cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.
Pathogen Testing
To protect mouse colonies from infection by pathogens and to assure that experimental preclinical data is not affected by such pathogens, all of Leinco’s Purified Functional PLATINUM™ antibodies are tested and guaranteed to be negative for all pathogens in the IDEXX IMPACT I Mouse Profile.
Storage and Handling
Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at ≤ -70°C. Avoid Repeated Freeze Thaw Cycles.
Country of Origin
USA
Shipping
Next Day 2-8°C
Applications and Recommended Usage?
Quality Tested by Leinco
FC The suggested concentration for clone 33D1 antibody for staining cells in flow cytometry is ≤ .25 μg per 106 cells in a volume of 100 μl or 100μl of whole blood. Titration of the reagent is recommended for optimal performance for each application.
Each investigator should determine their own optimal working dilution for specific applications. See directions on lot specific datasheets, as information may periodically change.

Description

Description

Specificity
Clone 33D1 recognizes mouse DCIR2.
Background
Dendritic cells are antigen presenting cells that have two functions. They scan the body collecting and processing antigen material that they present on the cell surface to T cells, and they maintain T cell tolerance to “self”. The morphology of dendritic cells is characterized by an extremely large surface-to-volume ratio. Murine splenic dendritic cells can occur in two types: myeloid (cDC) and lymphoid (pDC). Lymphoid dendritic cells produce high amounts of IFN-α and are also called Plasmacytoid dendritic cell because they have an appearance similar to plasma cells. Myeloid, or conventional dendritic cells, secrete IL-12, IL-6, TNF, and chemokines and can be further categorized into three subtypes (CD4−CD8+, CD4+CD8− and CD4−CD8−). These differ from other migratory dendritic cells such as Langerhans cells and interstitial dendritic cells that migrate from peripheral tissues to the lymph nodes. The exact nature and biological activity of the dendritic cell surface marker DCIR2 is currently unknown. DCs are known to play a role in several diseases including myeloid cancer, pDC leukemia, HIV, lupus erythematosus, Crohn's disease and ulcerative colitis. However, it is thought that DCs may be able to control cancer progression because increased densities of DC populations have been linked with better clinical outcome. Lung cancers have been found to include four different subsets of dendritic cells; some of which can activate immune cells that can suppress tumor growth. Dendritic cells have also been shown to play a role in the success of cancer immunotherapies in experimental models. Specifically, the immune checkpoint blocker anti-PD-1 has been shown to indirectly activate DCs through IFN-γ released from drug-activated T cells. Agonizing the non-canonical NF-κB pathway also activates DCs and further enhances anti-PD-1 therapy in an IL-12-dependent manner.
Antigen Distribution
Murine DCIR2 is found on dendritic cells of the thymus, spleen, lymph nodes, and Peyer’s patches. DCs in the bone marrow may express DCIR2 in the presence of GM-CSF. However, this expression is notably downregulated when IL-4 is present. Furthermore, DCIR2 has been found In vivo on brain dendritic cells post infection with T. gondii.
Function
GM-CSF is reported to increase expression of 33D1 antigen on dendritic cells from bone marrow cells and IL-4 reported to down regulate the 33D1 antigen.
Research Area
Immunology

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Clone 33D1 is most commonly used in vivo in mice to selectively deplete or track DCIR2+ conventional type 2 dendritic cells (cDC2), which are involved in immune regulation and disease models. This antibody specifically recognizes the DCIR2 (also called Clec4a4) surface marker, which is mainly expressed on cDC2 in mouse spleen, thymus, lymph nodes, Peyer's patches, and, under certain conditions, in the brain.

Typical in vivo applications of clone 33D1 include:

  • Dendritic cell (cDC2) depletion: Systemic administration of 33D1 (sometimes combined with complement) is used to ablate DCIR2+ DCs, allowing researchers to study the function of these cells in immune responses and tolerance, autoimmunity, and various disease models such as experimental autoimmune encephalomyelitis (EAE), diabetes, and cancer.
  • Cellular tracking and phenotyping: 33D1 can be injected into live mice to label and track cDC2 populations in tissues via flow cytometry or microscopy, providing insight into their migration, distribution, and participation during infection or inflammation.
  • Functional studies of antigen presentation: By depleting or labeling DCIR2+ dendritic cells in vivo, clone 33D1 enables experiments dissecting their specific roles in activating T cells, modulating T cell responses, and influencing the balance between immunity and tolerance.
  • Disease modeling: Manipulating the presence/function of cDC2 with 33D1 allows the study of their contributions to metabolic inflammation, tumor immunity, autoimmunity, and responses to pathogens.

Summary Table: Common In Vivo Uses of 33D1

ApplicationPurposeCore Contexts
Depletion of cDC2Study functional roles by removing these cellsAutoimmunity, infection, tumor models
Tracking and phenotypingLabeling cDC2 for distribution/migration studiesSteady state & inflammation response
Antigen presentation analysisDissecting impact on T cell priming and toleranceVaccination, immunotherapy, EAE

Key points

  • 33D1 is specific for mouse DCIR2/Clec4a4, which is mainly on cDC2.
  • In vivo, it is used for both depletion and tracking of cDC2.
  • Depletion aids in defining cDC2 roles in immunity and disease.
  • Used across mouse models of infection, cancer, autoimmunity, and tolerance.

Alternative applications include ex vivo sorting after in vivo antibody injection and synergy with various disease or inflammation models.

Commonly, the 33D1 antibody is used in combination with several other antibodies to refine the identification and isolation of dendritic cell (DC) subsets in mice by flow cytometry or other immunological techniques. The most frequently co-used antibodies and proteins include:

  • CD11c: A pan-dendritic cell marker frequently used to broadly identify dendritic cells.
  • CD8α: A specific marker for conventional dendritic cell subset 1 (cDC1), often used to distinguish between cDC1 and cDC2 (which express 33D1).
  • F4/80: A macrophage marker used for the exclusion of macrophages from dendritic cell gating strategies.
  • MHC class II (I-A/I-E): Used to confirm antigen-presenting cell (APC) identity and functionality.
  • B220 (CD45R): A marker for plasmacytoid dendritic cells (pDCs), used to exclude pDCs from analysis.
  • CD45: A pan-leukocyte marker, used for general gating of leukocytes in flow cytometry.

These markers collectively allow precise definition of cDC2 (33D1^+) cells within complex cell mixtures, such as splenocytes or lymph node suspensions.

Additional context and usage:

  • 33D1 is also referred to as DCIR2 or Clec4a4, and specifically marks cDC2, a dendritic cell subset important in antigen presentation and immunomodulation.
  • CD11c and 33D1 are often used together to distinguish cDC2 (CD11c^+33D1^+CD8α^-) from cDC1 (CD11c^+33D1^-CD8α^+).
  • F4/80 is critical for excluding macrophage populations, which may otherwise confound DC isolation.
  • MHC class II is essential for confirming that identified cells are competent antigen-presenting cells, not just surface marker positive myeloid cells.
  • B220 helps remove pDCs, which are functionally and phenotypically distinct from cDC2.

These marker combinations are standard in immunological studies and are recommended in many protocols for accurate dendritic cell subset identification in mouse tissues.

Clone 33D1 represents a landmark monoclonal antibody in dendritic cell research, with extensive scientific literature documenting its specificity, functionality, and applications in immunological studies. This rat-derived hybridoma antibody has become an essential tool for identifying and manipulating dendritic cell populations in mice.

Specificity and Antigen Recognition

Clone 33D1 recognizes a dendritic cell-specific surface antigen now identified as DCIR2 (Dendritic Cell Inhibitory Receptor 2), also known as Clec4a4 (C-type lectin domain family 4, member a4). The original characterization demonstrated that this antibody specifically killed 80-90% of dendritic cells from spleen and lymph node while showing no reactivity with other leukocytes, including Ia+ and Ia- macrophages, lymphocytes, granulocytes, platelets, or erythroid cells. Quantitative binding studies revealed that dendritic cells express approximately 14,000 binding sites per cell for this antibody.

The expression pattern is primarily observed in conventional type 2 dendritic cells (cDC2) within mouse thymus, spleen, lymph nodes, and Peyer's patches. An important technical consideration is that binding of clone 33D1 is calcium-dependent, requiring EDTA-free buffers containing Ca2+ ions for optimal staining.

Functional Applications

The antibody has proven invaluable for functional studies of dendritic cells through complement-mediated cytotoxicity. When used with rabbit complement, clone 33D1 selectively depletes dendritic cells from heterogeneous cell populations, enabling researchers to assess the functional consequences of dendritic cell removal. Studies using this approach demonstrated that removal of dendritic cells from unfractionated spleen suspensions reduced stimulatory capacity by 75-90% in mixed leukocyte reactions, comparable to results obtained with specific anti-Ia antibody and complement.

The antibody effectively ablates both proliferative and cytotoxic T cell responses when dendritic cells are eliminated, confirming that dendritic cells serve as the principal stimulators of primary immune responses. Importantly, the 33D1 antibody itself does not inhibit stimulation by enriched dendritic cell populations; cytotoxic effects require the presence of complement.

Biological Significance of DCIR2

Recent research has expanded understanding of the DCIR2 antigen recognized by clone 33D1. This protein is a 236 amino acid molecule with a predicted molecular weight of 27.3 kDa that undergoes post-translational modifications including disulfide bond formation, which may result in dimer appearance in immunoblot assays. DCIR2 is involved in multiple immunological processes including antigen recognition, suppression of autoimmunity through down-regulation of T cell priming, modulation of T cell responses, and progression of diet-induced obesity and inflammation.

Studies have shown that DCIR2-rich dendritic cells can ameliorate diseases with strong immune inflammatory components, including experimental autoimmune encephalomyelitis (EAE), experimental melanoma, and diabetes. The antibody's ability to target antigens to cDC2s was first demonstrated in 2007, when immunization with αDCIR2-OVA conjugates successfully induced immune responses.

Research Utility

Clone 33D1 has been extensively used for both in vitro and in vivo depletion of dendritic cells in experimental studies involving mouse models. Its stability as a marker is noteworthy—dendritic cells continue to express the 33D1 antigen after 4 days in culture, while macrophages and lymphocytes do not acquire it. This property, combined with its high specificity, makes clone 33D1 an essential tool for monitoring dendritic cell content in complex lymphoid mixtures and investigating dendritic cell biology in various disease contexts.

Dosing regimens for clone 33D1 (anti-mouse DCIR2) can vary significantly depending on the mouse model, experimental objectives, and immune context, but published protocols most frequently use intraperitoneal administration of 0.5 mg per mouse for 3 consecutive days, followed by weekly boosters up to 6 weeks in BALB/c models.

  • BALB/c mice (normal and AR models):

    • Standard regimen: 0.5 mg of purified anti-33D1 monoclonal antibody (mAb) via intraperitoneal (i.p.) injection for 3 successive days, supplemented with weekly boosters (same dose, 5 times) to maintain depletion for about 6 weeks.
    • Outcome: This regimen results in near-complete depletion of 33D1+ dendritic cells (DCs) in normal BALB/c mice for the duration, although some residual population may persist in allergen-sensitized (OVA/alum) mice, likely due to adjuvant or antigen-driven retention.
  • Adjuvant or sensitization effects:

    • Administration of OVA and alum as sensitizing agents in allergic airway models (AR) can partially prevent full eradication: after similar dosing (0.5 mg x3 days + weekly boosters), a small percentage of 33D1+ DCs remain. This suggests that inflammatory context or adjuvants may modulate depletion efficacy.
  • Other mouse strains/models:

    • While most published protocols focus on BALB/c models, dosing regimens for clone 33D1 may be adjusted for other strains (e.g., C57BL/6), body weight, immune status, or specific depletion goals. However, direct comparative dosing protocols for strains other than BALB/c are rarely specified in literature and may require empirical optimization based on pilot titration, target DC subset abundance, and readout timeline (e.g., flow cytometry after final dose).
  • Route of administration:

    • The most common and recommended route is intraperitoneal injection.

Summary Table: Common 33D1 Dosing Regimens

Mouse Model/ContextDose per MouseRouteScheduleNotes
BALB/c (normal)0.5 mgi.p.3 days + weekly boosters for 5 weeksDepletes splenic 33D1+ DCs for ~6 weeks
BALB/c (AR/OVA+alum)0.5 mgi.p.3 days + weekly boosters for 5 weeksPartial depletion; adjuvant preserves subset
Other strains/contextsVariablei.p.Empirical titration neededNo standard protocol; start with published dosing, optimize as required

Additional considerations:

  • Single-dose approaches (e.g., one-time 100 μg 33D1) have been used for acute experiments, but sustained depletion generally requires multiple or extended dosing.
  • Depletion efficacy should be validated by flow cytometry for each new protocol, especially when using different mouse strains or disease models.
  • Schedules may be further optimized based on the population kinetics of DCIR2+ cells (DC subset turnover) and experimental endpoints.

In summary, the most commonly cited regimen for clone 33D1 is 0.5 mg i.p. for 3 days plus weekly boosters, but efficacy and dose may require adjustment in models involving strong immune stimulation, different mouse strains, or specific research goals.

References & Citations

Steinman, R. M. et al. (1982) Pro. Natl. Acad. Sci. USA 79:161 Steinman, R. M. et al. (1983) J. Exp. Med. 157:613 Nussenzweig et al. 1982. Proc Natl Acad Sci U S A. 79(1):161-5. PMID: 6948298.
in vivo Protocol
General Western Blot Protocol

Certificate of Analysis

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Disclaimer AlertProducts are for research use only. Not for use in diagnostic or therapeutic procedures.