Recombinant Human ICAM-3

Recombinant Human ICAM-3 is valuable in research applications due to its critical roles in immune cell adhesion, activation, and signaling, making it a key tool for studying immune responses, cell-cell interactions, and disease mechanisms.

ICAM-3 (CD50) is a transmembrane glycoprotein expressed on all human leukocytes and serves as a primary ligand for the integrin LFA-1 (CD11a/CD18), facilitating adhesion between immune cells. This interaction is essential for the initiation of immune responses, particularly in the early stages of T cell activation by antigen-presenting cells (APCs) such as dendritic cells and Langerhans cells. ICAM-3 is especially important in the initial scanning and recognition of APCs by T cells, which is crucial for generating effective immune responses.

Key research applications include:

• Studying T cell activation and costimulation: ICAM-3 engagement provides a costimulatory signal that enhances T cell receptor (TCR)-mediated activation, proliferation, and cytokine production. It can lower the threshold for T cell activation, which is particularly relevant in models of immune aging or dysfunction.
• Modeling cell adhesion and migration: Recombinant ICAM-3 enables controlled investigation of leukocyte adhesion, migration, and transmigration processes, which are central to inflammation, immune surveillance, and tissue repair.
• Investigating apoptosis and phagocytosis: ICAM-3 mediates the clearance of apoptotic cells by attracting macrophages, making it useful for studying mechanisms of cell death and immune clearance.
• Cancer research: ICAM-3 is implicated in cancer cell migration, epithelial-mesenchymal transition, and stemness via signaling pathways such as PI3K/AKT, making recombinant ICAM-3 relevant for exploring tumor progression and metastasis.
• Viral infection models: ICAM-3 costimulation enhances HIV-1 transcription and replication in CD4+ T cells, providing a model for studying viral pathogenesis and immune modulation.

Experimental uses of recombinant ICAM-3 include:

• Bioassays to measure cell adhesion, migration, and activation.
• ELISA standards for quantifying soluble ICAM-3 in biological samples.
• Functional studies of immune cell interactions and signaling pathways.

In summary, recombinant human ICAM-3 is a versatile reagent for dissecting immune cell interactions, signaling mechanisms, and disease processes, particularly in immunology, oncology, and virology research.

Yes, recombinant human ICAM-3 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to your assay system. Most commercial ICAM-3 ELISA kits use recombinant human ICAM-3 as the standard for generating the calibration curve, and these standards are specifically formulated and validated for accurate quantification.

Key considerations:

• Standard Curve Preparation: ELISA protocols require a standard curve generated from known concentrations of ICAM-3, typically using recombinant protein. Serial dilutions of the recombinant standard are prepared to cover the assay’s detection range (e.g., 0.02–20 ng/mL, 78–5000 pg/mL, or as specified by your kit).
• Assay Compatibility: Ensure the recombinant ICAM-3 you use is compatible with the antibodies in your ELISA. Most kits are designed to detect both natural and recombinant forms, but the recombinant standard should match the epitope recognized by the capture and detection antibodies.
• Validation: If you are using recombinant ICAM-3 from a source different from your ELISA kit, validate its performance by comparing its standard curve to that provided by the kit, ensuring similar sensitivity and linearity.
• Research Use Only: Commercial ELISA kits and recombinant standards are typically for research use only and not for diagnostic or therapeutic applications.

Protocol Example:

• Reconstitute the recombinant ICAM-3 standard as directed.
• Prepare serial dilutions to generate a standard curve (e.g., 20, 10, 5, 2.5, 1.25, 0.625 ng/mL).
• Run standards in parallel with your samples.
• Use the standard curve to interpolate ICAM-3 concentrations in unknown samples.

Summary Table: Recombinant ICAM-3 as ELISA Standard

In summary: Recombinant human ICAM-3 is the standard of choice for quantitative ELISA calibration, but always ensure compatibility and validation with your specific assay system.

Recombinant Human ICAM-3 has been validated for several key applications in published research, primarily in bioassays, ELISA (as a standard), and studies of cell adhesion, immune regulation, and cancer biology.

Validated Applications:

• Bioassays:
  Recombinant ICAM-3 is used to study cell adhesion, particularly its interaction with leukocyte integrins such as LFA-1 (CD11a/CD18). Immobilized ICAM-3 supports the adhesion of PMA-stimulated human peripheral blood cells, making it a standard tool for functional assays of immune cell interactions.

• ELISA (Standard):
  Soluble ICAM-3 is used as a standard in ELISA to quantify circulating ICAM-3 levels, especially in studies of autoimmune diseases and juvenile rheumatoid arthritis.

• Cell Migration and Apoptosis Studies:
  ICAM-3 has been used in research on cancer cell migration, stemness, and apoptosis, particularly in lung and breast cancer cell lines. It is involved in the regulation of signaling pathways such as PI3K/AKT and is a target for anti-inflammatory and anti-metastatic drug studies.

• Macrophage Chemoattraction and Tethering:
  Apoptotic cell-derived ICAM-3 promotes macrophage recruitment and tethering to apoptotic cells, relevant for studies of immune clearance and inflammation.

• Biomarker Research:
  ICAM-3 expression is investigated as a biomarker for radiation resistance in cervical cancer and as a diagnostic marker in various cancers and autoimmune conditions.

• Cell Adhesion Mechanism Studies:
  Used to dissect the molecular mechanisms of cell adhesion in human immunity, especially where ICAM-3 function is distinct from ICAM-1 and ICAM-2.

Additional Context:

• ICAM-3 is not present in mouse models, so recombinant human ICAM-3 is essential for human-specific studies.
• It is commonly used in in vitro systems, including whole cell assays and protein interaction studies.
• Recombinant ICAM-3 is also used to validate antibody specificity in Western blot and flow cytometry, though these are applications of the protein as an immunogen rather than direct functional assays.

Summary Table of Validated Applications

These applications are supported by peer-reviewed publications and are widely used in immunology, oncology, and cell biology research.

To reconstitute and prepare Recombinant Human ICAM-3 protein for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Reconstitute the protein in sterile distilled or deionized water. A typical concentration range is 0.1–1.0 mg/mL; some protocols recommend at least 100 μg/mL.
• Avoid vigorous mixing: Do not vortex or pipette forcefully, as this can denature the protein. Instead, gently swirl or invert the vial to dissolve the protein.
• Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.
• If a lower working concentration is needed, dilute the reconstituted stock with sterile PBS (pH 7.4) or cell culture medium as appropriate for your assay.
• For cell culture applications, ensure the final buffer is compatible with your cells (e.g., PBS or serum-free medium). If the protein was lyophilized from PBS, reconstitution in water will restore the original buffer; otherwise, adjust as needed.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage: After reconstitution, store at 2–8 °C for up to one week, or at –20 °C to –80 °C for longer-term storage. Avoid repeated freeze-thaw cycles.

Optional for stability: Some protocols recommend adding 5–50% glycerol to the reconstituted protein for enhanced stability, especially if storing at –20 °C.

Summary Table: Recombinant Human ICAM-3 Reconstitution

Note: Always consult the specific product datasheet for any unique instructions related to your recombinant ICAM-3 preparation, as formulations and tags may vary between suppliers. Optimal working concentrations for cell culture should be determined empirically for your specific assay.