Recombinant Mouse MCP-5

Recombinant Mouse MCP-5 (CCL12) is a potent chemokine used in research to study immune cell recruitment, inflammation, and host responses to infection, particularly in murine models. Its primary function is to attract monocytes, eosinophils, and lymphocytes, but not neutrophils, making it highly relevant for dissecting mechanisms of monocyte-driven inflammation and allergic responses.

Key scientific applications and rationale for use:

• Chemotaxis Assays: MCP-5 is a robust chemoattractant for monocytes and lymphocytes, enabling quantitative analysis of cell migration in vitro and in vivo.
• Receptor Signaling Studies: MCP-5 signals through the CCR2 receptor, which is preferentially expressed on monocytes and activated lymphocytes. This allows for detailed investigation of CCR2-mediated pathways and their role in immune cell trafficking.
• Inflammation and Allergy Models: MCP-5 expression is upregulated in murine models of pulmonary inflammation and allergic challenge, making recombinant MCP-5 valuable for mimicking or modulating these responses in experimental systems.
• Host-Pathogen Interaction: MCP-5 is involved in the host response to pathogens, such as during infection with Nippostrongylus brasiliensis, providing a tool to study immune cell recruitment during infection.
• Functional Homology: MCP-5 is structurally and functionally homologous to human MCP-1 (CCL2), allowing for comparative studies between mouse and human chemokine systems.

Best practices for use:

• Employ recombinant MCP-5 in dose-dependent chemotaxis assays to quantify monocyte and lymphocyte migration.
• Use in cell signaling experiments to assess CCR2 activation, such as calcium flux assays.
• Integrate into murine models of inflammation or infection to study leukocyte recruitment and cytokine networks.
• Combine with neutralizing antibodies or receptor antagonists to dissect MCP-5-specific effects.

In summary, Recombinant Mouse MCP-5 is essential for research focused on monocyte biology, chemokine signaling, and immune cell migration in mouse models, providing mechanistic insights into inflammation, allergy, and infection.

Yes, recombinant mouse MCP-5 (CCL12) can be used as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is purified and its concentration is accurately determined. Most commercial ELISA kits for mouse MCP-5 (CCL12) include a recombinant mouse MCP-5 standard specifically for this purpose, and the assay is designed to recognize both natural and recombinant forms of the protein.

Key Points:

• Recombinant Protein Suitability: Recombinant mouse MCP-5 is suitable for use as an ELISA standard because the assay antibodies are typically developed to recognize both natural and recombinant forms of the protein.
• Purification and Concentration: The recombinant protein should be purified, and its concentration should be measured accurately (e.g., by HPLC or other validated methods) to ensure reliable quantification.
• Kit Compatibility: Many ELISA kits (such as those from Invitrogen, R&D Systems, Novus Biologicals, and others) explicitly state that their standards are recombinant mouse MCP-5 and are intended for use in generating a standard curve for quantification.
• Carrier Protein: If the recombinant MCP-5 is supplied with a carrier protein (such as BSA), this is generally acceptable for ELISA standards. Carrier-free versions are also available and may be preferred for certain applications.

Recommendations:

• Always follow the manufacturer's instructions for reconstitution and dilution of the recombinant standard.
• Prepare a serial dilution of the recombinant MCP-5 to generate a standard curve that spans the expected concentration range of your samples.
• Use fresh pipette tips for each dilution and repeat in duplicate or triplicate for accuracy.
• Construct a new standard curve for every plate and each experiment to ensure reliable quantification.

In summary, recombinant mouse MCP-5 is a valid and commonly used standard for ELISA quantification, as long as it is properly purified and its concentration is accurately known.

Recombinant Mouse MCP-5 (CCL12) has been validated for several key applications in published research, primarily in the context of immunology and inflammation studies. The most commonly validated applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant Mouse MCP-5 is widely used as a standard or analyte in ELISA protocols to quantify MCP-5 levels in biological samples such as cell culture supernatants, cell lysates, serum, and plasma. This application is crucial for monitoring MCP-5 expression in various disease models, including asthma and rheumatoid arthritis.

• Cell/Tissue Culture Functional Assays: MCP-5 is used in cell migration and chemotaxis assays to study its role in attracting eosinophils, monocytes, and lymphocytes, and to investigate its signaling through the CCR2 receptor. These assays help elucidate MCP-5’s function in allergic inflammation and immune cell recruitment.

• Western Blot: Recombinant MCP-5 has been validated for use as a control or standard in Western blotting to detect MCP-5 protein expression in tissue or cell extracts.

• Immunohistochemistry (IHC) and Immunofluorescence (IF): MCP-5 is used in IHC and IF protocols to localize and visualize MCP-5 protein in tissue sections, aiding in the study of its distribution during inflammatory responses.

• Immunoprecipitation (IP) and Flow Cytometry: These applications utilize recombinant MCP-5 to study protein-protein interactions and to quantify MCP-5-expressing cells, respectively.

• Functional Studies: MCP-5 is employed in functional assays to assess its biological activity, such as chemotactic potency and cytokine induction in immune cells.

Additional Context:

• MCP-5 has been implicated in models of asthma, rheumatoid arthritis, and other inflammatory diseases, where its expression and function are studied using the above applications.
• In knockout mouse models, MCP-5 levels are measured to investigate compensatory mechanisms and its role in monocyte recruitment and inflammation.

Summary Table of Validated Applications

These applications are supported by published research and product validation data, making recombinant Mouse MCP-5 a versatile tool for immunological and inflammation-related studies.

To reconstitute Recombinant Mouse MCP-5 (CCL12) protein for cell culture experiments, dissolve the lyophilized protein in sterile water or sterile PBS to a concentration of 100 µg/mL or higher. After reconstitution, gently mix the solution and aliquot for storage to avoid repeated freeze-thaw cycles.

Step-by-step protocol:

• Preparation:

  • Briefly centrifuge the vial to collect the powder at the bottom before opening.
  • Use sterile technique throughout to prevent contamination.

• Reconstitution:

  • Add sterile distilled water, sterile PBS, or 0.1% BSA in water to achieve a final concentration of 100 µg/mL to 0.5 mg/mL (e.g., add 100 µL to 100 µg protein for 1 mg/mL).
  • Gently mix by pipetting or inversion; avoid vigorous vortexing to prevent protein denaturation.
  • If using a carrier protein (such as BSA), this can help stabilize the protein for long-term storage.

• Storage:

  • Store the reconstituted protein at 4°C for short-term use (2–7 days).
  • For long-term storage, aliquot and freeze at –20°C to –80°C.
  • Avoid repeated freeze-thaw cycles, as this can degrade the protein.

• Dilution for cell culture:

  • Before adding to cell culture, dilute the stock solution in cell culture medium or buffer to the desired working concentration (e.g., ED50 for chemotaxis is 0.06–0.6 ng/mL for Baf3-mCCR2 cells).
  • If possible, add a carrier protein (e.g., 0.1% BSA) to working solutions to further stabilize the protein.

Additional notes:

• Always consult the specific product datasheet for recommended diluents and concentrations, as formulation may vary by supplier.
• If the protein is supplied with a carrier (e.g., BSA), reconstitute as directed in PBS; if carrier-free, use sterile water or PBS.
• Document all preparation steps and aliquot sizes for reproducibility.

Summary Table:

This protocol ensures optimal activity and stability of recombinant MCP-5 for cell culture applications.