DCSTAMP Antibody

What is DC-STAMP and why is it important in research?

DC-STAMP is a seven-pass-transmembrane receptor-like protein that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. It plays an essential role in:

• Cell-to-cell fusion during osteoclastogenesis (bone resorption processes)

• Regulating immune cell interactions and inflammatory responses

• Modulating cytokine production in dendritic cells and macrophages

Research significance: DC-STAMP knockout mice exhibit mild osteopetrosis and impaired osteoclast fusion, indicating its importance in bone homeostasis . DC-STAMP also regulates inflammatory cytokine production, making it relevant to both bone and immunological research .

What are the major types of DC-STAMP antibodies available for research?

How can researchers validate the specificity of DC-STAMP antibodies?

Validation should include multiple complementary approaches:

• Genetic validation: Compare antibody binding between wild-type and DC-STAMP knockout samples. For example, Western blotting analysis with anti-DC-STAMP antibody 1A2 detected DC-STAMP in thymus and spleen lysates from wild-type mice but showed greatly reduced signals in Dcstamp-knockout mice .

• Recombinant protein validation: Test antibody recognition of recombinant DC-STAMP. The 1A2 antibody showed specific binding to DC-STAMP in PTHR-DC-STAMP fusion construct-transfected RAW cells .

• Immunoprecipitation-Western blot: Confirm antibody specificity by immunoprecipitating DC-STAMP followed by detection via Western blot. Monomeric (~53 kDa) and dimeric (~106 kDa) forms can be detected under denaturing and non-denaturing conditions respectively .

• Cell type comparison: Compare staining patterns across different DC-STAMP-expressing cell populations (e.g., CD14+CD16- vs. CD14+CD16+ monocytes) .

Flow Cytometry

• Sample preparation: For surface staining, block Fc receptors with anti-CD16/CD32 for 15 minutes on ice before adding fluorophore-conjugated anti-DC-STAMP antibody (30 minutes, 4% FCS/PBS) .

• Controls: Include 7-AAD for viability gating. For proper gating, use unstained, isotype control, and single-stained controls .

• Antibody recommendations: 1A2 clone detects DC-STAMP with 100-fold higher sensitivity compared to KR104 in flow cytometry applications .

• Intracellular staining: Use Cytofix/Cytoperm kit for permeabilization when assessing total (not just surface) DC-STAMP .

Western Blotting

• Membrane extraction: Use native membrane protein extraction methods for optimal results, especially when studying transmembrane proteins like DC-STAMP .

• Non-denaturing vs. denaturing conditions: Under non-denaturing conditions, dimeric DC-STAMP (~106 kDa) is detected; under denaturing conditions, monomeric form (~53 kDa) is detected .

• Antibody dilution: Start with 1:1000 dilution in 5% BSA/TBST for overnight incubation at 4°C .

Immunoprecipitation

• Protocol: Use EZView Red Protein A Affinity Gel beads after incubating membrane proteins with anti-DC-STAMP antibody for 1 hour at 4°C .

• Detection: Follow with SDS-PAGE and immunoblotting using the same or different anti-DC-STAMP antibody .

How should researchers quantify DC-STAMP expression in flow cytometry experiments?

For accurate quantification:

• Baseline comparisons: Compare mean fluorescence intensity (MFI) across experimental conditions. Note that CD14+CD16+ monocytes show higher DC-STAMP expression (MFI: 2748) compared to CD14+CD16- monocytes (MFI: 1747) .

• Controls for accurate measurement:

  • Include unstained cells (e.g., MFI: 317)

  • Isotype control antibody (e.g., MFI: 715)

  • Single-color controls for compensation

• Analysis considerations:

  • Gate on viable cells (using 7-AAD exclusion)

  • Consider cell size/complexity effects on fluorescence intensity

  • Report relative MFI compared to control populations

How can DC-STAMP antibodies be used to study osteoclastogenesis?

DC-STAMP antibodies provide valuable tools for investigating osteoclast development:

• Functional blocking: The 1A2 antibody inhibits osteoclast precursor (OCP) fusion in vitro, making it useful for studying the mechanism of multinucleation during osteoclastogenesis .

• Expression kinetics: DC-STAMP surface expression shows reciprocal regulation with CD16 during osteoclastogenesis. While CD16 expression increases over time, DC-STAMP levels decline .

• Cellular localization: Use fluorescence microscopy with rhodamine phalloidin (for actin), FITC-conjugated anti-DC-STAMP 1A2, and DAPI (for nuclei) to visualize DC-STAMP distribution during cell fusion:

  • Fix cells in cold methanol (-20°C, 10 minutes)

  • Permeabilize with 0.1% saponin/0.2% BSA/PBS (15 minutes)

  • Stain with rhodamine phalloidin, FITC-anti-DC-STAMP, and DAPI (2 hours, room temperature)

  • Mount in 90% glycerol/10% 1M Tris (pH8)

• Expression analysis: Track DC-STAMP mRNA expression during osteoclast differentiation using qPCR with the following primer sets:

  • DC-STAMP: Commercial qPCR Primer Assay

  • β-actin (control): Forward 5′-AGATGTGGATCAGCAAGCAG-3′, Reverse 5′-GCGCAAGTTAGGTTTTGTCA-3′

What is the role of DC-STAMP in inflammatory processes, and how can antibodies help study this?

DC-STAMP bridges bone resorption and inflammation through several mechanisms:

• Cytokine regulation: DC-STAMP knockout significantly alters cytokine production profiles. DC-STAMP knockdown mBMDCs secrete less IL-6, IL-12, TNF-α, and IL-10 while producing more IL-1 .

• Transcriptional effects: Research shows DC-STAMP affects cytokine gene transcription:

  • IL-6 and IL-12p40 mRNA levels decrease by 20-75% and 20-65% respectively in DC-STAMP knockdown cells

  • IL-1α and IL-1β mRNA levels increase by 25-48% and 27-35% respectively

• Inflammatory model studies: In the Tg(hTNF) arthritis mouse model, DC-STAMP knockout shows:

  • Decreased inflammatory cell accumulation in synovium

  • Lower frequency of CCL2+ and F4/80+ macrophages

  • Reduced CD3+ T cells in knee synovium

  • Decreased serum levels of murine TNF

• Macrophage phenotype: DC-STAMP regulates macrophage polarization. DC-STAMP-deficient macrophages show impaired Tnf, Il1β, and Inos expression and reduced production of IL1β, TNF, IL6, and CCL2 .

What are the key technical considerations when working with DC-STAMP dimers versus monomers?

The dimeric structure of DC-STAMP presents unique experimental challenges:

• Sample preparation:

  • For detecting dimers (~106 kDa): Use native membrane protein extraction methods and non-denaturing conditions .

  • For monomers (~53 kDa): Use standard denaturing conditions with SDS-PAGE .

• Antibody selection:

  • The 1A2 monoclonal antibody efficiently detects DC-STAMP dimers in flow cytometry .

  • Optimize antibody concentration based on application (1:1000 for Western blot, 1:100 for immunoprecipitation) .

• Detection strategy:

  • Flow cytometry: More efficient for detecting surface dimers on live cells

  • Western blot: Better for distinguishing between monomeric and dimeric forms

  • Cross-linking experiments: May help stabilize dimers for analysis

• Controls and validation:

  • Include both wild-type and DC-STAMP knockout samples

  • Compare results across multiple antibodies (e.g., 1A2 and KR104)

  • Include positive controls from known DC-STAMP-expressing tissues like thymus and spleen

How does the ITIM motif in DC-STAMP influence its signaling properties and experimental approaches?

The immunoreceptor tyrosine-based inhibitory motif (ITIM) in DC-STAMP's cytoplasmic domain (S407FYPSV412) provides important signaling capabilities:

• Signaling mechanism: The ITIM motif likely counteracts immunoreceptor tyrosine-based activation motif (ITAM) signaling from molecules like CD16. This suggests DC-STAMP may provide inhibitory counterbalance to activation signals .

• Experimental approaches:

  • Phosphorylation studies: Use anti-phosphotyrosine mAb 4G10 to detect phosphorylated ITIM following immunoprecipitation with anti-DC-STAMP 1A2

  • Co-immunoprecipitation: Identify binding partners using anti-DC-STAMP 1A2 pulldown followed by mass spectrometry or Western blotting

  • Mutational analysis: Create ITIM-mutant DC-STAMP constructs to assess functional consequences

• Technical considerations:

  • Use CytoBuster Protein Extraction Reagent for cell lysis

  • Perform immunoprecipitation with anti-DC-STAMP 1A2

  • Probe blots with anti-phosphotyrosine antibodies

  • Develop with SuperSignal West Pico or Femto chemiluminescent substrate

What are common issues when using DC-STAMP antibodies and how can they be resolved?

How should researchers interpret seemingly contradictory results from DC-STAMP experiments?

Several factors may contribute to contradictory findings:

• Age-dependent effects: DC-STAMP knockout mice develop different phenotypes with age. Young mice show mild osteopetrosis, while aged mice develop systemic autoimmunity with lymphoproliferation, splenomegaly, and autoantibodies .

• Model-specific differences: The suppression of inflammation in Dcstamp-/-;Tg(hTNF) model contrasts with autoimmunity in aged Dcstamp-/- mice, suggesting context-dependent functions .

• Cell type variations: DC-STAMP knockdown in bone marrow dendritic cells impairs IL-6, IL-12, and TNF release while increasing IL-1β, promoting Th2 differentiation .

• Technical variations: Different antibodies (1A2 vs. KR104) target distinct epitopes and show varying efficacy in different applications. For example, KR104 targets the 1st extracellular domain and fails to recognize PTHR-DC-STAMP fusion protein, while 1A2 targets the 4th extracellular domain .

• Experimental interpretation: When findings conflict, consider:

  • Comparing antibody specificity and epitope locations

  • Validating with multiple detection methods

  • Confirming knockout/knockdown efficiency

  • Considering developmental stage and cell type

How might DC-STAMP antibodies contribute to understanding bone disorders and inflammatory diseases?

DC-STAMP stands at the intersection of bone homeostasis and inflammation, offering promising research avenues:

• Biomarker development: DC-STAMP may serve as an activity biomarker in inflammatory arthritis, as knockout mice show decreased local and systemic inflammation in arthritis models .

• Therapeutic targeting: Anti-DC-STAMP antibodies like 1A2 that block osteoclast formation could potentially be developed into therapeutic agents for conditions with excessive bone resorption .

• Mechanistic studies: DC-STAMP antibodies can help elucidate:

  • How DC-STAMP modulates both bone resorption and inflammation

  • The currently unknown ligand(s) of DC-STAMP

  • The role of DC-STAMP in different inflammatory arthritis subtypes

• Clinical correlations: Measuring DC-STAMP expression on peripheral blood monocytes using flow cytometry could potentially correlate with disease activity in conditions like rheumatoid arthritis or psoriatic arthritis .

What technical innovations might improve DC-STAMP detection and functional analysis?

Future methodological improvements could include:

• Single-cell analysis: Combining DC-STAMP antibody labeling with single-cell RNA sequencing to correlate DC-STAMP expression with transcriptional profiles of osteoclast precursors or inflammatory cells.

• Advanced imaging: Super-resolution microscopy with fluorescently-labeled DC-STAMP antibodies to visualize molecular distribution during cell fusion events.

• Proximity labeling: Using DC-STAMP antibodies conjugated to proximity labeling enzymes (BioID, APEX2) to identify proximal interacting partners in living cells.

• CRISPR screening: Combining DC-STAMP antibody-based phenotyping with CRISPR screens to identify genes that modulate DC-STAMP expression, dimerization, or signaling.

• Structural studies: Using antibody fragments to facilitate crystallization and structural determination of DC-STAMP, particularly given the technical challenges in membrane protein structural studies highlighted in search result .