CD63 Monoclonal Antibody

What is CD63 and what cellular functions does it regulate?

CD63 (also known as LAMP-3, TSPAN30, Granulophysin) is a tetraspanin superfamily protein that appears as a 25 kDa protein when deglycosylated but typically migrates as a 50-60 kDa molecule due to heavy glycosylation . It functions as a cell surface receptor for TIMP1 and plays crucial roles in cellular signaling cascades. CD63 activates ITGB1 and integrin signaling pathways, leading to activation of AKT, FAK/PTK2, and MAP kinases . Through these pathways, CD63 promotes cell survival, reorganization of the actin cytoskeleton, cell adhesion, spreading, and migration . Additionally, CD63 contributes significantly to intercellular communication by facilitating the transfer of proteins, lipids, and RNA between cells through its role in exosome formation .

What are the primary applications of CD63 monoclonal antibodies in research?

CD63 monoclonal antibodies serve multiple critical research applications:

• Immunocytochemistry (ICC): Visualizes CD63 subcellular localization in fixed cells

• Flow Cytometry: Quantifies CD63 expression on cell surfaces or intracellularly

• Immunohistochemistry (IHC-P): Detects CD63 in paraffin-embedded tissue sections

• Western Blotting (WB): Identifies CD63 protein expression levels in cell lysates

• Exosome Research: Isolates and characterizes exosomes from complex biological samples

• Functional Studies: Investigates CD63's role in mast cell degranulation and allergic reactions

• Cell Adhesion Analysis: Examines CD63's interaction with extracellular matrix proteins

In which tissue and cell types is CD63 expression most relevant for research?

CD63 exhibits a broad expression pattern across multiple cell and tissue types:

CD63 primarily resides in late endocytic vesicles under resting conditions but translocates to the plasma membrane following cell activation, making it a valuable activation marker .

What fixation and permeabilization methods yield optimal results for CD63 detection?

The choice of fixation and permeabilization method significantly impacts CD63 detection:

For Immunocytochemistry:

• Paraformaldehyde (4%) fixation for 10-15 minutes at room temperature yields optimal results

• Permeabilization with saponin preserves membrane structure while allowing antibody access to intracellular CD63

• A549 human lung carcinoma cells show specific cytoplasmic staining when fixed with this approach

For Flow Cytometry:

• For surface CD63: Minimal fixation without permeabilization is recommended

• For total CD63: Paraformaldehyde fixation followed by saponin permeabilization effectively detects both surface and intracellular pools

• Human PBMC monocytes show distinct CD63 staining patterns using this approach

For Immunohistochemistry:

• Formalin-fixed, paraffin-embedded (FFPE) tissues typically require antigen retrieval methods

• Human spleen tissue shows strong CD63 positivity following appropriate antigen retrieval

What controls are essential when validating CD63 antibody specificity?

Rigorous control experiments are crucial for ensuring CD63 antibody specificity:

• Isotype Controls: Include appropriate isotype-matched antibodies to assess non-specific binding

• Positive Control Tissues/Cells: Human PBMCs and spleen tissue serve as reliable positive controls

• Blocking Peptides: Synthetic peptides derived from CD63 sequence can confirm specificity through competition assays

• Cross-Reactivity Testing: Verify species specificity, as antibodies like 12A10 and 7A6 recognize rat CD63 but not human or mouse CD63

• Multiple Detection Methods: Confirm findings across different techniques (flow cytometry, ICC, WB) to validate specificity

How should researchers address variable glycosylation when detecting CD63?

CD63 appears as a smear at 50-60 kDa in immunoblots due to extensive glycosylation, which complicates detection and quantification:

• Deglycosylation Treatment: Enzymatic treatment with PNGase F produces a sharper band at approximately 25 kDa

• Recognition Requirements: Some antibodies recognize CD63 only in its non-reduced form, indicating conformation-dependent epitopes

• Multiple Antibody Approach: Using antibodies targeting different epitopes can help overcome glycosylation-related detection issues

• Sample Preparation: For consistent results, standardize protein extraction methods to minimize variability in glycosylation patterns

How do CD63 antibodies affect mast cell function and allergic responses?

CD63 monoclonal antibodies demonstrate significant effects on mast cell function through several mechanisms:

• Selective Inhibition of Degranulation: Anti-CD63 antibodies potently suppress FcɛRI-induced degranulation in mast cells without affecting leukotriene synthesis

• Adhesion-Dependent Effects: The inhibitory effect is observed only in adherent cells, not in suspension cells, revealing a critical link between adhesion and degranulation pathways

• Substrate Specificity: Anti-CD63 inhibits degranulation in cells adhering to fibronectin and vitronectin but not in cells on fibrinogen, correlating with its differential effects on adhesion to these substrates

• Signaling Pathway Selectivity: Anti-CD63 does not affect FcɛRI-induced global tyrosine phosphorylation or calcium mobilization but specifically impairs the Gab2-PI3K pathway essential for both degranulation and adhesion

• In Vivo Efficacy: Anti-CD63 antibodies inhibit FcɛRI-mediated allergic reactions in animal models, suggesting therapeutic potential

What is the relationship between CD63 and integrin signaling in cell adhesion?

CD63 functions as a critical regulator of integrin-mediated adhesion through several mechanisms:

• Direct Integrin Interaction: CD63 physically interacts with α3, α4, and α6 chains of β1 integrins, modulating their activity

• Comparative Inhibition Effects: Anti-CD63-mediated inhibition of adhesion to fibronectin is comparable to combined anti-α5 and anti-β1 antibodies, while anti-CD63 inhibition of adhesion to vitronectin exceeds that of anti-β3 antibodies

• Substrate-Specific Modulation: CD63 antibodies inhibit cell adhesion to fibronectin and vitronectin (which enhance degranulation) but not to fibrinogen (which does not affect degranulation)

• Signaling Pathway Integration: Through integrin interactions, CD63 activates downstream signaling involving AKT, FAK/PTK2, and MAP kinases that promote cell survival, cytoskeletal reorganization, and migration

• Functional Correlation: The inhibitory effects of anti-CD63 on degranulation correlate precisely with its effects on adhesion, suggesting a mechanistic link between these processes

What technical challenges exist in CD63-based exosome isolation and characterization?

Researchers face several technical challenges when using CD63 for exosome research:

• Heterogeneous Expression: CD63 expression varies significantly between exosome subpopulations, potentially leading to biased isolation

• Co-isolation of Contaminants: Extracellular vesicle preparations often contain both free proteins and lipoproteins that must be removed for accurate analysis

• Epitope Accessibility: Heavy glycosylation of CD63 can mask epitopes, affecting antibody binding efficiency

• Multi-marker Requirement: Comprehensive exosome characterization requires combining CD63 with other tetraspanins (CD9, CD81) and exosome markers

• Standardization Challenges: Variable isolation methods yield inconsistent results, necessitating standardized protocols for reliable quantification

Recent methodological improvements include:

• Improved isolation techniques that effectively remove both free proteins and lipoproteins

• Development of immunogold single extracellular vesicular RNA and protein (Au SERP) biochips for more precise characterization

• Framework approaches for rapid comparison of different extracellular vesicle isolation methods

How can researchers address inconsistent CD63 staining patterns between experiments?

When facing variable CD63 staining results, researchers should systematically evaluate:

• Antibody Clone Selection: Different clones (such as MEM-259, 460305, BGG-3) recognize distinct epitopes with varying accessibility across sample types

• Protocol Optimization by Cell Type:

  • For A549 cells: 25 μg/mL antibody concentration, 3-hour room temperature incubation yields optimal cytoplasmic staining

  • For PBMC monocytes: Permeabilization is critical for detecting intracellular CD63 pools

  • For tissues: Antigen retrieval methods must be optimized based on fixation protocol

• Sample Preparation Variables:

  • Cell detachment method affects epitope preservation (EDTA preferred over trypsin)

  • Cell activation state dramatically influences CD63 expression and localization

  • Fixation duration and temperature require optimization for each cell type

• Technical Validation Approaches:

  • Implement dilution series to determine optimal antibody concentration

  • Include positive control cells with known high CD63 expression

  • Document all experimental variables (cell density, passage number, etc.)

What storage and handling considerations ensure optimal CD63 antibody performance?

To maintain antibody functionality over time:

• Long-term Storage: Store at -20°C for up to one year

• Working Stock Management: For frequent use, store at 4°C for up to one month

• Avoid Repeated Freeze-Thaw: Aliquot antibodies to minimize freeze-thaw cycles

• Reconstitution Protocol: Restore with deionized water for optimal reconstitution

• Buffer Composition: Most CD63 antibodies perform optimally in phosphate-buffered saline with 150mM NaCl, 0.02% sodium azide, and 50% glycerol with 0.4-0.5mg/ml BSA

How can researchers differentiate between surface and intracellular CD63 pools?

CD63 distributes between the plasma membrane and intracellular compartments, particularly late endosomes and lysosomes. To distinguish between these pools:

• For Flow Cytometry:

  • Surface CD63: Stain non-permeabilized cells at 4°C to prevent internalization

  • Total CD63: Fix cells with paraformaldehyde and permeabilize with saponin before staining

  • Calculate the ratio of surface to total CD63 to quantify distribution

• For Immunofluorescence Microscopy:

  • Surface CD63: Stain live cells at 4°C before fixation

  • Total CD63: Fix and permeabilize before staining

  • Use confocal microscopy to visualize distinct subcellular compartments

• For Biochemical Analysis:

  • Surface Biotinylation: Selectively label and isolate surface proteins

  • Subcellular Fractionation: Separate membrane fractions from endosomal/lysosomal fractions

  • Western Blotting: Compare CD63 levels in different fractions

What role does CD63 play in cancer research and potential therapeutic applications?

CD63 has emerging significance in cancer research:

• Biomarker Potential: CD63 expression in A549 lung carcinoma cells and other cancer cell lines suggests utility as a diagnostic or prognostic marker

• Exosome-Based Liquid Biopsies: CD63-positive exosomes in patient fluids may serve as non-invasive cancer biomarkers

• Immunotherapy Response Prediction: Emerging evidence suggests CD63-positive extracellular vesicles may predict responses to immunotherapy in non-small cell lung cancer patients

• Therapeutic Targeting: The ability of anti-CD63 antibodies to modulate cellular functions suggests potential for therapeutic development

• Drug Delivery Systems: CD63's role in exosome biology makes it a candidate for targeted drug delivery approaches

How can CD63 antibodies advance understanding of neurodegenerative diseases?

While not explicitly covered in the provided search results, CD63's roles in vesicular trafficking and intercellular communication have significant implications for neurodegenerative disease research:

• Exosome-Mediated Protein Transfer: CD63-positive exosomes may contribute to the spread of pathogenic proteins in diseases like Alzheimer's and Parkinson's

• Neuroinflammatory Mechanisms: CD63's role in immune cell function relates to neuroinflammatory processes in neurodegenerative conditions

• Biomarker Development: CD63-positive exosomes in cerebrospinal fluid or blood may serve as biomarkers of disease progression

• Therapeutic Intervention: Modulating CD63 function might affect exosome-mediated transfer of pathogenic proteins between neurons