CD62E Antibody

What is CD62E and what is its significance in inflammation research?

CD62E, also known as E-selectin or endothelial-leukocyte adhesion molecule-1 (ELAM-1), is a 97-115 kDa member of the selectin family . This adhesion molecule is expressed by endothelial cells upon stimulation with pro-inflammatory cytokines including TNFalpha and IL-1beta . CD62E plays a critical role in mediating leukocyte rolling and the initial interaction of neutrophils with endothelium during inflammatory conditions . Unlike other cellular adhesion molecules, CD62E is minimally expressed by unstimulated endothelium and is specifically upregulated in response to inflammatory cytokines, making it an excellent marker for activated endothelium in inflammation research . Its involvement in leukocyte extravasation during inflammation and potential role in tumor cell metastasis makes it a significant target for immunological and oncological research .

How does CD62E expression differ between resting and activated endothelial cells?

In resting endothelial cells, CD62E is minimally expressed, with baseline expression levels often below the detection threshold of standard analytical techniques . Following stimulation with inflammatory cytokines such as TNFalpha or IL-1beta, endothelial cells rapidly upregulate surface CD62E expression . This inducible expression pattern makes CD62E particularly valuable as a biomarker of endothelial activation. Flow cytometric analysis has demonstrated this dramatic difference, with Human Umbilical Vein Endothelial Cells (HUVECs) showing minimal CD62E expression at rest but significant expression after 6 hours of stimulation with 25 ng/mL of recombinant human TNF-alpha . This pattern of expression is consistent across different vascular beds, though the magnitude and kinetics of induction may vary depending on the tissue origin of the endothelial cells and the specific inflammatory stimulus applied .

What are the optimal conditions for detecting CD62E expression in flow cytometry experiments?

For optimal detection of CD62E expression by flow cytometry, several methodological considerations are essential:

• Cell Preparation: Human Umbilical Vein Endothelial Cells (HUVECs) are commonly used as they reliably express CD62E upon activation . Cells should be stimulated with TNFalpha (typically 25 ng/mL) for approximately 6 hours to induce robust CD62E expression .

• Antibody Selection: Several validated monoclonal antibodies are available, including:

  • P2H3 clone: Can be used at 0.5-1 μg per test (10^5 to 10^8 cells in 100 μL)

  • 68-5H11 clone: Pre-diluted for use with 1 × 10^6 cells in a 100-μL experimental sample

  • BBIG-E4 clone: Particularly useful for both flow cytometry and functional studies

• Flow Cytometer Settings: For PE-conjugated antibodies, use excitation at 488-561 nm and emission at 578 nm with blue, green, or yellow-green lasers . APC-conjugated antibodies can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser .

• Controls: Include unstimulated endothelial cells as negative controls and properly matched isotype controls (e.g., MAB002) to set gates accurately .

• Staining Protocol: Follow manufacturer-recommended protocols, typically involving incubation with the primary antibody followed by secondary antibody if using non-conjugated primaries . For conjugated antibodies, direct staining according to the manufacturer's protocol is sufficient .

How should researchers optimize immunohistochemical detection of CD62E in tissue sections?

For optimal immunohistochemical detection of CD62E in tissue sections, researchers should follow these methodological guidelines:

• Tissue Preparation: For paraffin-embedded sections, heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic is essential for unmasking CD62E epitopes . Fresh frozen sections may require different fixation protocols, typically with acetone or paraformaldehyde.

• Antibody Selection and Concentration: The BBIG-E4 and P2H3 clones have been validated for immunohistochemistry . Optimal concentration is typically 10 μg/mL, though titration is recommended for each application and tissue type .

• Incubation Conditions: For paraffin sections, overnight incubation at 4°C with the primary antibody yields optimal results . For frozen sections, 3-hour incubation at room temperature is often sufficient .

• Detection Systems: HRP-DAB Cell & Tissue Staining Kit (brown) with hematoxylin counterstaining (blue) provides excellent contrast for visualizing CD62E-positive endothelial cells . Fluorescent detection can be achieved with appropriate secondary antibodies, such as NorthernLights™ 557-conjugated Anti-Mouse IgG, with DAPI counterstaining for nuclear visualization .

• Positive Controls: Inflamed tissues or TNFα-activated cultured endothelial cells should be included as positive controls to verify staining specificity .

• Negative Controls: Include sections stained with isotype-matched control antibodies and tissues known to lack CD62E expression as negative controls .

How can CD62E antibodies be employed in functional assays to study leukocyte-endothelial interactions?

CD62E antibodies offer powerful tools for studying leukocyte-endothelial interactions through various functional assays:

• Adhesion Inhibition Assays: Several CD62E antibodies, including the P2H3 clone, have been demonstrated to inhibit cellular adhesion to cytokine-activated endothelial cells . This property can be leveraged to quantify the specific contribution of CD62E to leukocyte adhesion by measuring the reduction in adherent cells when CD62E is blocked. The 68-5H11 clone specifically inhibits the adhesion between endothelial CD62E molecules and carbohydrate ligands on neutrophils .

• Flow-Based Adhesion Assays: Researchers can use parallel plate flow chambers or microfluidic devices coated with activated endothelial cells to study leukocyte rolling and adhesion under physiologically relevant shear stress conditions. CD62E antibodies can be applied to specifically block this interaction, allowing for precise quantification of CD62E's contribution to the multi-step adhesion cascade .

• Transmigration Assays: Transwell systems with activated endothelial cell monolayers can be used to study leukocyte transmigration. Pre-treatment with CD62E antibodies helps determine the role of this adhesion molecule in facilitating not just adhesion but also subsequent transmigration .

• In vivo Imaging: Fluorescently labeled CD62E antibodies can be used in intravital microscopy studies to visualize CD62E expression on activated endothelium in live animals, providing spatial and temporal information about CD62E upregulation during inflammation .

• Knockout Validation: Results obtained with CD62E antibody blocking should be validated against genetic approaches (siRNA knockdown or CRISPR/Cas9 knockout) to confirm specificity and rule out potential off-target effects of antibody binding .

What role does CD62E play in pathological conditions beyond acute inflammation?

CD62E's functions extend beyond acute inflammation to several critical pathological processes:

• Transplant Vasculopathy (TV): Recent research has identified CD62E as a key mediator in the development of transplant vasculopathy. Anti-HLA-I antibody-mediated changes in CD62E expression in endothelial cells contribute to leukocyte recruitment and subsequent intima hyperplasia . Heme oxygenase-1 (HO-1) has been identified as a modulator of CD62E-dependent endothelial cell functions, suggesting potential therapeutic approaches to prevent transplant rejection .

• Cancer Metastasis: CD62E plays a role in the metastasis of certain tumor cells, facilitating their interaction with endothelium during hematogenous spread . The mechanism appears to involve specific glycosylated ligands on cancer cells that bind to CD62E on activated endothelium.

• Chronic Inflammatory Diseases: In conditions like rheumatoid arthritis, inflammatory bowel disease, and psoriasis, persistent upregulation of CD62E contributes to ongoing leukocyte recruitment and tissue damage. CD62E antibodies have been used experimentally to study these processes and evaluate potential therapeutic approaches .

• Atherosclerosis: CD62E expression on arterial endothelium contributes to monocyte recruitment to developing atherosclerotic plaques. Blocking CD62E has been explored as a strategy to reduce atherosclerotic progression in experimental models .

• Ischemia-Reperfusion Injury: During reperfusion following ischemic events, rapid upregulation of CD62E contributes to neutrophil recruitment and subsequent tissue damage. Anti-CD62E approaches have shown promise in reducing this inflammatory response in experimental models .

What are common pitfalls in CD62E detection and how can they be overcome?

Researchers frequently encounter several challenges when detecting CD62E expression:

• Low Basal Expression: The minimal expression of CD62E on resting endothelium often leads to false-negative results. Solution: Always include positive controls (TNFα-activated endothelial cells) to verify assay functionality .

• Temporal Expression Dynamics: CD62E expression follows specific kinetics after stimulation, typically peaking at 4-6 hours and declining thereafter. Solution: Perform time-course experiments to identify optimal detection windows for your specific experimental system .

• Cell Culture Conditions: Passage number and culture conditions can significantly affect endothelial cell responsiveness. Solution: Use low-passage endothelial cells (ideally <P6) and validate cytokine responsiveness before critical experiments .

• Antibody Clone Specificity: Different antibody clones recognize distinct epitopes, potentially yielding different results. Solution: Validate findings with multiple antibody clones when possible, and select clones based on the specific application requirements .

• Tissue Fixation Effects: Overfixation can mask CD62E epitopes. Solution: Optimize fixation protocols for each tissue type and application, and employ appropriate antigen retrieval methods for formalin-fixed tissues .

• Flow Cytometry Sensitivity: CD62E detection requires optimized cytometer settings. Solution: Carefully titrate antibodies, use appropriate fluorophores based on your cytometer configuration, and include fluorescence-minus-one (FMO) controls .

• Enzymatic Dissociation Effects: Enzymatic dissociation of tissues or endothelial monolayers can cleave surface CD62E. Solution: Use gentle dissociation methods and minimize processing time between cell collection and analysis .

How should researchers standardize CD62E antibody applications across different experimental platforms?

Standardization of CD62E antibody applications requires systematic approach:

• Antibody Validation: Before conducting extensive experiments, validate each antibody lot using:

  • Flow cytometry on TNFα-stimulated versus unstimulated HUVECs

  • Western blot to confirm specific binding to the expected 97-115 kDa protein

  • Immunofluorescence on activated endothelial cells with appropriate controls

• Titration for Each Application: Optimal antibody concentration varies by application:

  • Flow cytometry: Typically 0.5-1 μg per 10^5-10^8 cells in 100 μL

  • Immunohistochemistry: Generally 10 μg/mL, but requires optimization

  • Western blot: Start with 1:1000 dilution and adjust as needed

• Standardized Activation Protocols: For consistent CD62E induction:

  • Use recombinant human TNFα at 25 ng/mL for 6 hours as a standard stimulus

  • Document lot numbers and sources of all cytokines used

  • Include time-course experiments to identify peak expression times

• Reference Standards: Establish internal reference samples:

  • Cryopreserved aliquots of activated endothelial cells

  • Standardized positive control lysates for Western blotting

  • Well-characterized tissue sections for immunohistochemistry

• Reporting Guidelines: Document and report:

  • Antibody clone, manufacturer, lot number, and concentration

  • Detailed activation protocols (cytokine concentration, time, temperature)

  • Complete staining protocols with all buffer compositions

  • Instrument settings for flow cytometry or imaging parameters

How is CD62E research contributing to our understanding of inflammatory disease mechanisms?

Recent advances in CD62E research are shedding new light on inflammatory disease mechanisms:

• Organ-Specific Endothelial Responses: Emerging evidence suggests that CD62E expression and function vary across different vascular beds, contributing to organ-specific patterns of leukocyte recruitment during inflammation . This heterogeneity helps explain why certain inflammatory diseases preferentially affect specific organs.

• Transplant Vasculopathy Mechanisms: Research has identified CD62E as a crucial mediator in transplant vasculopathy, with HO-1 modulation emerging as a promising therapeutic approach to prevent leukocyte recruitment and subsequent intima hyperplasia . This finding connects CD62E function directly to transplant outcomes and organ failure.

• Post-Translational Modifications: Studies of CD62E glycosylation patterns are revealing how these modifications affect binding specificity for different leukocyte subsets, potentially explaining the selective recruitment of specific immune cell populations in different inflammatory contexts .

• Microenvironmental Regulation: Beyond cytokine stimulation, factors such as shear stress, oxygen tension, and metabolic conditions are now recognized as important modulators of CD62E expression and function, adding complexity to our understanding of endothelial activation in vivo .

• Chronic Inflammation Maintenance: CD62E's contribution to establishing and maintaining chronic inflammatory states is being investigated, particularly its role in the transition from acute to chronic inflammation through sustained leukocyte recruitment and activation .

What novel methodological approaches are being developed for studying CD62E-dependent processes?

Cutting-edge methodological approaches are advancing CD62E research:

• Single-Cell Analysis: Flow cytometry and single-cell RNA sequencing are revealing heterogeneity in CD62E expression among endothelial cells within the same vessel, suggesting functional specialization of individual cells in mediating leukocyte recruitment .

• Advanced Imaging Techniques: Super-resolution microscopy and intravital multiphoton imaging are providing unprecedented spatial and temporal resolution of CD62E-mediated leukocyte-endothelial interactions in vivo, revealing dynamic aspects previously unobservable with conventional techniques .

• Microfluidic Organ-on-Chip Models: These platforms recreate physiologically relevant microenvironments for studying CD62E-dependent leukocyte adhesion under flow conditions while allowing precise control over variables such as shear stress, oxygen levels, and cytokine gradients .

• CRISPR-Based Functional Genomics: High-throughput CRISPR screens are identifying novel regulators of CD62E expression and function, complementing traditional antibody-based approaches with genetic manipulation strategies .

• Computational Modeling: Systems biology approaches are integrating experimental data into mathematical models that predict how CD62E expression patterns and binding kinetics influence leukocyte recruitment dynamics under various inflammatory conditions .

• Antibody Engineering: Development of bispecific antibodies targeting CD62E and other endothelial adhesion molecules is enabling more precise manipulation of specific steps in the leukocyte adhesion cascade, offering new tools for both research and potential therapeutic applications .