Recombinant Mouse ICAM-1 Fc Chimera

Using Recombinant Mouse ICAM-1 Fc Chimera in research offers several advantages for studying cell adhesion, immune cell interactions, and inflammatory processes, particularly in mouse models. This fusion protein combines the extracellular domain of mouse ICAM-1 (CD54) with the Fc region of an immunoglobulin, enhancing stability, ease of purification, and functional versatility.

Key scientific applications and benefits include:

• Cell Adhesion Assays: Immobilized recombinant mouse ICAM-1 Fc chimera supports the adhesion of activated leukocytes (e.g., PMA-stimulated HSB2 or HL60 cells), making it a robust tool for quantifying integrin-mediated cell adhesion and dissecting the molecular mechanisms of immune cell trafficking.
• Immunology and Inflammation Research: ICAM-1 is a critical ligand for LFA-1 (CD11a/CD18) on leukocytes, mediating immune cell adhesion, migration, and signaling. The chimera is widely used to model these interactions in vitro, enabling studies on T cell activation, migration, and positive selection in the thymus, as well as neutrophil infiltration and tissue injury in inflammation models.
• Bioassays and Functional Studies: The Fc fusion format allows for easy immobilization on assay plates or beads, facilitating high-throughput screening, flow cytometry, and mechanistic studies of cell-cell interactions.
• Receptor-Ligand Binding Studies: The recombinant chimera can be used to probe the specificity and affinity of integrin-ICAM-1 interactions, including cross-species binding (e.g., mouse LFA-1 with mouse ICAM-1), and to block or compete for endogenous ICAM-1 function in experimental systems.
• Enhanced Stability and Detection: The Fc region increases the protein’s half-life and enables detection or capture using anti-Fc antibodies, simplifying experimental workflows and improving reproducibility.
• Versatility in Experimental Design: The protein is suitable for a range of applications, including ELISA, flow cytometry, cell culture, and in vivo models, supporting studies in immunology, oncology, and vascular biology.

In summary, Recombinant Mouse ICAM-1 Fc Chimera is a versatile and reliable reagent for dissecting the molecular basis of immune cell adhesion, migration, and signaling in mouse systems, with broad utility in both basic and translational research.

Yes, you can use Recombinant Mouse ICAM-1 Fc Chimera as a standard for quantification or calibration in ELISA assays, provided that the ELISA kit is designed to detect mouse ICAM-1 and the chimera is compatible with the detection antibodies and assay format.

Key Considerations:

• Specificity: The recombinant protein must be recognized by the capture and detection antibodies used in your ELISA kit. Most commercial kits are validated for both natural and recombinant forms of ICAM-1, including Fc chimeras.
• Standard Curve: Prepare a dilution series of the recombinant protein in the same buffer as your samples to generate a standard curve. This allows for accurate quantification of ICAM-1 in your samples.
• Carrier Protein: If the recombinant protein is supplied with a carrier (e.g., BSA), ensure that the carrier does not interfere with the ELISA. For most kits, carrier-free versions are preferred for use as standards.
• Validation: It is good practice to validate the standard by comparing it with a known standard provided by the kit manufacturer, if available.

References:

• R&D Systems and BioLegend both provide recombinant mouse ICAM-1 Fc chimera proteins that are suitable for use as standards in ELISA assays.
• Thermo Fisher Scientific and Sigma-Aldrich also offer ELISA kits that recognize both natural and recombinant forms of ICAM-1.

By following these guidelines, you can effectively use Recombinant Mouse ICAM-1 Fc Chimera as a standard for your ELISA assays.

Recombinant Mouse ICAM-1 Fc Chimera has been validated for a range of applications in published research, primarily in studies involving cell adhesion, immune cell migration, and molecular interaction assays.

Key validated applications include:

• Bioassay: Extensively used to study T cell activation, migration, and adhesion, as well as in functional assays involving immune cell interactions and signaling pathways.
• Adhesion Assay: Used to assess cell adhesion properties, particularly leukocyte or lymphocyte binding to ICAM-1 under static or flow conditions.
• Binding Assay: Applied to quantify or characterize the binding of integrins (such as LFA-1) or other ligands to ICAM-1.
• Cell Culture: Used as a substrate or supplement to mimic endothelial ICAM-1 in vitro, supporting studies of immune cell behavior and migration.
• Flow Cytometry: Utilized as a reagent to detect or block ICAM-1 interactions on cell surfaces.
• Surface Plasmon Resonance (SPR): Employed to measure real-time binding kinetics between ICAM-1 and its ligands, such as LFA-1.
• Control: Used as a positive or negative control in various immunological and biochemical assays.

Representative published research applications:

• T cell migration and activation: Used in studies dissecting the role of ICAM-1 in T cell homing, adhesion, and signaling, including microfluidic tumor vasculature models and thymocyte selection assays.
• Autoimmune and inflammatory models: Applied in research on experimental autoimmune encephalomyelitis, ischemia-reperfusion injury, and leukocyte trafficking.
• Single-cell force measurements: Used in biomembrane force probe assays to analyze mechanical aspects of cell adhesion.
• Substrate for expansion of antigen-specific T cells: Combined with chemokines and cytokines to enhance T cell proliferation in vitro.

Summary Table of Validated Applications

These applications are well-supported by published literature and product validation data, making the recombinant mouse ICAM-1 Fc chimera a versatile tool for immunological and cell biology research.

To reconstitute and prepare Recombinant Mouse ICAM-1 Fc Chimera protein for cell culture experiments, follow these steps based on standard protocols and manufacturer recommendations:

Reconstitution

1. Reconstitution Buffer:
   Reconstitute the lyophilized protein in sterile phosphate-buffered saline (PBS) at a concentration of 0.4 mg/mL (400 µg/mL).

   • Example: For a vial containing 100 µg of protein, add 250 µL of sterile PBS.

2. Procedure:

   • Gently swirl the vial to dissolve the protein. Avoid vigorous shaking to prevent denaturation.
   • Allow the protein to dissolve completely at room temperature.

Preparation for Cell Culture (Coating Plates)

1. Coating Solution:
   Dilute the reconstituted protein in sterile PBS to a working concentration of 12.5 µg/mL.

2. Coating Protocol:

   • Add 100 µL of the diluted protein solution per well of a 96-well plate.
   • Incubate the plate at 4°C overnight (or at 37°C for 1–2 hours if a shorter incubation is preferred).
   • After incubation, remove the solution and wash the wells once with sterile PBS.

3. Blocking (Optional but Recommended):

   • Block nonspecific binding sites by adding 150–200 µL of blocking buffer (e.g., 1% BSA in PBS) per well.
   • Incubate for 1 hour at 37°C.
   • Wash the wells once with PBS before adding cells.

Cell Adhesion Assay

• Add 5 × 10⁴ cells/well to the coated plate.
• Incubate at 37°C for 1 hour.
• Wash away non-adherent cells and proceed with downstream analysis.

Storage

• After reconstitution: Store at 2–8°C for up to 1 month or at –20 to –70°C for up to 3 months under sterile conditions.
• Avoid repeated freeze-thaw cycles.

Notes

• Optimal coating concentration and incubation time may vary depending on your cell type and experimental setup. Always perform a pilot experiment to optimize conditions.
• Ensure all solutions and plates are sterile for cell culture applications.

These steps will help you prepare the Recombinant Mouse ICAM-1 Fc Chimera protein for reliable cell adhesion and culture experiments.