CLE10 Antibody

What is CLEC10A and why is it important in immunological research?

CLEC10A, also known as macrophage galactose/N-acetyl-galactosamine (GalNAc) specific lectin (MGL), CD301, DC-ASGPR, and HML, is a 40 kDa type II transmembrane glycoprotein belonging to the C-type lectin family. It consists of a 39 amino acid cytoplasmic region, a 21 amino acid transmembrane segment, and a 256 amino acid extracellular domain with one carbohydrate recognition domain (CRD) and a neck region . CLEC10A is particularly significant in immunological research because it is expressed on immature myeloid dendritic cells and alternatively activated (tolerogenic) macrophages, making it an important marker for studying immune tolerance mechanisms and dendritic cell function . Its expression is notably upregulated by immunosuppressants like dexamethasone, suggesting a role in modulating immune responses .

What are the known splice variants of CLEC10A and how might they affect antibody selection?

Alternate splicing generates multiple isoforms of human CLEC10A/MGL with 27 amino acid, 3 amino acid, and/or 4 amino acid deletions within the extracellular domain (ECD) . These variations can significantly impact antibody binding specificity. When selecting antibodies for research, it's crucial to consider which epitopes might be affected by these deletions. For optimal results, researchers should select antibodies that target conserved regions present across all relevant isoforms of interest or choose isoform-specific antibodies if studying particular variants. Cross-reactivity testing with known isoforms is recommended prior to experimental application to ensure appropriate detection of the target CLEC10A variants in your specific cell types or tissues.

How do human and murine CLEC10A homologs differ, and what are the implications for antibody cross-reactivity?

Unlike humans and rats that carry a single gene for CLEC10A/MGL, mice have two closely related genes: MGL1 and MGL2. Within the carbohydrate recognition domain (CRD), human CLEC10A/MGL shares 64-70% amino acid sequence identity with mouse MGL1, mouse MGL2, and rat MGL . This moderate sequence conservation has significant implications for antibody selection. Some antibodies, like the goat anti-human/mouse CLEC10A antibody (Catalog # AF4888), are specifically designed to recognize both human and mouse variants . When studying across species, researchers should carefully validate cross-reactivity and may need species-specific antibodies for certain applications. The functional differences are also notable - human CLEC10A and mouse MGL2 exhibit similar ligand preferences (binding terminal nonsialylated alpha- or beta-linked GalNAc moieties), while mouse MGL1 has different binding characteristics .

What are the optimal detection methods for CLEC10A in different cell types?

The optimal detection method for CLEC10A varies depending on the cell type and research question. For immature myeloid dendritic cells and alternatively activated macrophages, flow cytometry using fluorophore-conjugated antibodies (like Alexa Fluor® 350-conjugated antibodies) provides excellent sensitivity and allows for multiparameter analysis . When performing flow cytometry on human dendritic cells, a typical protocol involves staining with 10 μL/10^6 cells of anti-CLEC10A antibody followed by appropriate secondary antibodies if using non-conjugated primary antibodies . For protein expression analysis, Western blot under reducing conditions using PVDF membranes probed with 1 μg/mL of anti-CLEC10A antibody has been successfully demonstrated with human dendritic cell lysates, detecting CLEC10A at approximately 35 kDa . For tissue sections, immunohistochemistry should be optimized with appropriate antigen retrieval techniques, with particular attention to fixation methods as these can affect the carbohydrate recognition domain structure.

What controls should be included when validating CLEC10A antibody specificity?

Proper validation of CLEC10A antibody specificity requires multiple controls. First, include isotype controls matched to the primary antibody's host species and immunoglobulin class (e.g., Catalog # AB-108-C as demonstrated in flow cytometry experiments) . Second, incorporate positive controls using cells known to express CLEC10A (immature dendritic cells) and negative controls (cells that don't express CLEC10A). For advanced validation, consider using CLEC10A knockdown or knockout cells to confirm specificity. Cross-reactivity testing with related C-type lectins is also recommended due to structural similarities within this family. When using secondary detection systems, include controls omitting the primary antibody to assess non-specific binding. For Western blot applications, pre-absorption of the antibody with recombinant CLEC10A protein can further confirm specificity by abolishing true positive signals.

How can researchers effectively detect CLEC10A in complex tissue samples?

Detecting CLEC10A in complex tissue samples requires a multifaceted approach. For immunohistochemistry or immunofluorescence, antigen retrieval optimization is critical since CLEC10A is a glycoprotein and improper retrieval can destroy epitopes. A recommended protocol includes using citrate buffer (pH 6.0) with heat-induced epitope retrieval, followed by blocking with serum from the species in which the secondary antibody was raised. For enhanced specificity in tissues with high background, tyramide signal amplification can improve signal-to-noise ratio. For mass spectrometry-based detection, immunoprecipitation using validated anti-CLEC10A antibodies followed by targeted proteomic analysis can provide high specificity. In single-cell analysis applications, combining anti-CLEC10A antibodies with antibodies against CD11c, CD14, and other myeloid markers enables precise identification of CLEC10A-expressing cell subpopulations within heterogeneous tissue microenvironments.

How can CLEC10A antibodies be utilized to study tolerogenic dendritic cell function?

CLEC10A antibodies provide valuable tools for investigating tolerogenic dendritic cell (tDC) function through multiple sophisticated approaches. First, researchers can use fluorophore-conjugated CLEC10A antibodies to isolate and purify CLEC10A-high tDCs by fluorescence-activated cell sorting (FACS) for functional studies. Second, antibody-mediated crosslinking of CLEC10A can be employed to study downstream signaling pathways - this approach involves plate-bound or bead-coupled anti-CLEC10A antibodies to engage CLEC10A receptors on dendritic cells, followed by phosphoproteomic analysis to map signaling cascades. Third, blocking antibodies against CLEC10A can reveal its role in tDC-T cell interactions by disrupting the binding between CLEC10A on tDCs and carbohydrate determinants on CD45 (RA, RB, and RC isoforms) expressed by T cells . This interaction normally inhibits effector T cell activation and induces apoptosis, making it a critical regulatory mechanism . For advanced in vivo applications, researchers can use fluorescently-labeled CLEC10A antibodies for intravital microscopy to track tDC migration and function in living tissues during immune response development.

What approaches can be used to investigate CLEC10A's role in pathogen recognition and entry?

Investigating CLEC10A's role in pathogen recognition and entry requires sophisticated experimental approaches. CLEC10A has been shown to bind GP envelope glycoproteins on Marburg and Ebola viruses, enhancing viral entry and infectivity . To study these interactions, researchers can employ neutralizing anti-CLEC10A antibodies in viral infection assays using susceptible cell types. By pre-treating cells with these antibodies before viral challenge, researchers can quantify changes in infection rates. For mechanistic studies, competitive binding assays using anti-CLEC10A antibodies and viral envelope proteins can identify specific binding epitopes. Advanced imaging techniques like super-resolution microscopy combined with dual-labeled CLEC10A antibodies and viral proteins allow visualization of receptor clustering during viral attachment. For in vivo applications, humanized mouse models treated with anti-CLEC10A antibodies before viral challenge can elucidate the receptor's role in pathogenesis. Additionally, researchers can develop antibody-based strategies to block CLEC10A-pathogen interactions as potential therapeutic approaches against viruses that exploit this receptor for cellular entry.

How do CLEC10A's carbohydrate binding properties influence antibody epitope accessibility?

CLEC10A's function as a carbohydrate recognition receptor creates unique considerations for antibody epitope accessibility. CLEC10A selectively binds terminal nonsialylated alpha- or beta-linked GalNAc moieties on O-linked carbohydrates, including the Tn carcinoma antigen . This ligand-binding activity can potentially mask antibody epitopes in or near the carbohydrate recognition domain (CRD). When designing experiments, researchers should consider that ligand-bound CLEC10A may display altered epitope accessibility compared to unbound receptor. To address this challenge, consider using antibodies targeting multiple distinct epitopes, particularly those recognizing regions outside the CRD. For applications requiring detection of ligand-bound receptor, develop specialized protocols incorporating carbohydrate competition assays to confirm antibody binding is unaffected by ligand engagement. Researchers investigating CLEC10A-ligand interactions should test antibody binding under both ligand-saturated and ligand-free conditions to fully characterize potential interference. For structural biology applications, epitope mapping studies using hydrogen-deuterium exchange mass spectrometry (HDX-MS) in the presence and absence of carbohydrate ligands can identify antibodies whose binding is unaffected by receptor occupation status.

What factors contribute to inconsistent CLEC10A antibody staining in flow cytometry?

Inconsistent CLEC10A antibody staining in flow cytometry can result from multiple technical and biological factors. From a technical perspective, inadequate blocking of Fc receptors on myeloid cells is a common issue - implement comprehensive blocking with appropriate sera (2-5% for 15-30 minutes) before antibody staining . Cell preparation methods can affect epitope integrity; optimize fixation procedures (if required) using low concentrations (0.5-1%) of paraformaldehyde to preserve CLEC10A structure while maintaining cell integrity. From a biological standpoint, CLEC10A expression is dynamically regulated by factors like dexamethasone and activation state . Standardize cell culture conditions and activation protocols when comparing CLEC10A expression across experiments. Another potential source of variability is CLEC10A's internalization upon ligand binding - minimize time between sample preparation and analysis to reduce artificial downregulation. If working with clinical samples, patient heterogeneity and treatment status can significantly impact CLEC10A expression. Finally, antibody lot-to-lot variation can introduce inconsistency - validate new antibody lots against previously tested lots using positive control samples before conducting critical experiments.

How can researchers distinguish between specific and non-specific binding in CLEC10A Western blot applications?

Distinguishing between specific and non-specific binding in CLEC10A Western blots requires rigorous controls and optimization strategies. As demonstrated in validated protocols, CLEC10A appears at approximately 35 kDa under reducing conditions on PVDF membranes when probing human dendritic cell lysates . To minimize non-specific binding, optimize blocking conditions (5% non-fat dry milk or 3-5% BSA) and antibody concentrations (starting with 1 μg/mL for anti-CLEC10A antibodies) . Include positive controls (lysates from immature dendritic cells) and negative controls (cell types known not to express CLEC10A). For definitive validation, consider using lysates from CLEC10A knockout or knockdown cells alongside wild-type samples. Pre-adsorption controls, where the antibody is pre-incubated with recombinant CLEC10A protein before Western blotting, can confirm specificity - true CLEC10A bands should disappear in pre-adsorbed samples. Multiple antibodies targeting different CLEC10A epitopes should recognize the same bands, providing additional confirmation. When analyzing multiple spliced variants, carefully compare observed molecular weights with theoretical predictions for each isoform, noting that post-translational modifications (particularly glycosylation) can affect migration patterns.

What confounding factors should researchers consider when interpreting CLEC10A expression data?

When interpreting CLEC10A expression data, researchers must consider several potential confounding factors. First, CLEC10A expression is highly sensitive to cellular activation states - expression levels increase significantly during dendritic cell maturation and in alternatively activated (tolerogenic) macrophages . Second, treatment with immunosuppressants like dexamethasone upregulates CLEC10A expression, requiring careful documentation of any treatments applied to experimental systems . Third, CLEC10A undergoes dynamic subcellular trafficking upon ligand binding, potentially affecting detection depending on the method used. Fourth, the presence of multiple splice variants means that antibodies targeting different epitopes may detect different subsets of total CLEC10A expression . Fifth, post-translational modifications, particularly glycosylation patterns, can vary between cell types and activation states, potentially affecting antibody binding. Sixth, when comparing expression across species, remember that mice have two CLEC10A homologs (MGL1 and MGL2) with different ligand preferences . Finally, sample preparation methods can significantly impact detection - membrane proteins like CLEC10A require appropriate extraction buffers and handling to maintain structural integrity and epitope accessibility.

How can CLEC10A antibodies be used to investigate interactions with tumor-associated carbohydrate antigens?

CLEC10A's ability to bind tumor-associated carbohydrate antigens (TACAs), particularly the Tn antigen (GalNAc-α-O-Ser/Thr), presents significant opportunities for cancer research . Researchers can employ anti-CLEC10A antibodies to investigate these interactions through several sophisticated approaches. Dual-color confocal microscopy using fluorescently-labeled anti-CLEC10A antibodies and anti-Tn antibodies can visualize co-localization at the tumor-immune cell interface. For functional studies, blocking antibodies against CLEC10A can reveal its role in dendritic cell recognition and processing of tumor antigens by disrupting CLEC10A-TACA binding. Flow cytometry-based binding assays comparing wild-type and enzymatically deglycosylated tumor cells can quantify CLEC10A-dependent interactions, using anti-CLEC10A antibodies to detect receptor expression on immune cells. For translational applications, researchers can explore targeted therapeutic strategies by developing bispecific antibodies that link CLEC10A-expressing dendritic cells to tumor cells displaying Tn antigens, potentially enhancing antigen presentation and anti-tumor immunity. Additionally, anti-CLEC10A antibodies can be used to monitor changes in receptor expression and distribution during tumor progression and in response to immunotherapies.

What methodological approaches can differentiate between the functions of CLEC10A and related C-type lectin receptors?

Differentiating between CLEC10A and related C-type lectin receptors requires sophisticated methodological approaches. First, researchers should employ highly specific antibodies validated against multiple C-type lectins to ensure no cross-reactivity. When designing knockout or knockdown studies, careful sequence analysis must confirm target specificity to avoid off-target effects on related receptors. For functional differentiation, researchers can compare binding profiles using glycan arrays - while CLEC10A selectively binds nonsialylated alpha- or beta-linked GalNAc moieties, other C-type lectins recognize different carbohydrate structures . To distinguish receptor-specific signaling pathways, phosphoproteomic analysis following receptor-specific antibody engagement can map downstream events unique to each receptor. For in vivo studies, conditional knockout systems targeting specific receptors in defined cell populations can reveal non-redundant functions. Single-cell RNA sequencing combined with protein detection using validated antibodies can identify cells co-expressing multiple C-type lectins and reveal receptor-specific gene expression signatures. Finally, structural biology approaches utilizing receptor-specific antibodies for co-crystallization can identify unique binding pocket features that distinguish CLEC10A from related receptors.

How might CLEC10A antibodies be utilized in studying immune tolerance mechanisms?

CLEC10A antibodies offer valuable tools for investigating immune tolerance mechanisms, particularly given CLEC10A's expression on tolerogenic dendritic cells and its inhibitory effects on T cell activation . Researchers can employ anti-CLEC10A antibodies to identify and isolate tolerogenic dendritic cell populations for detailed functional characterization. Since CLEC10A expressed on tolerogenic dendritic cells binds carbohydrate determinants on CD45 (RA, RB, and RC but not RO isoforms) on T, NK, and B cells, researchers can use blocking antibodies to disrupt this interaction and assess its impact on immune suppression mechanisms . Time-lapse confocal microscopy with fluorescently-labeled anti-CLEC10A antibodies can visualize receptor clustering and internalization during tolerogenic dendritic cell-T cell interactions. For in vivo applications, anti-CLEC10A antibodies can be used to track receptor-expressing cells in models of transplantation tolerance, autoimmunity, and cancer immune evasion. To explore therapeutic applications, researchers can investigate whether targeting CLEC10A with agonistic or antagonistic antibodies modulates tolerance induction. Additionally, combining CLEC10A detection with functional assays measuring T cell proliferation, cytokine production, and apoptosis can establish correlations between receptor expression levels and tolerogenic function under different physiological and pathological conditions.