Recombinant Mouse MIP-3α

Recombinant Mouse MIP-3α (CCL20) is widely used in research applications to study immune cell recruitment, enhance vaccine efficacy, and model inflammatory responses due to its potent chemotactic activity for immature dendritic cells and lymphocytes.

Key scientific applications and rationale:

• Targeting Immature Dendritic Cells (iDCs): MIP-3α binds to CCR6, a receptor highly expressed on iDCs, facilitating their recruitment to sites of antigen presentation. This property is exploited in vaccine research, where fusion of MIP-3α to antigens enhances delivery to iDCs, resulting in improved adaptive immune responses and increased antibody production.

• Enhancing Vaccine Immunogenicity: Incorporating MIP-3α into vaccine constructs has been shown to significantly increase both humoral (antibody) and cellular (T-cell) responses in mouse models. For example, vaccines fused to MIP-3α elicit higher and more sustained antibody titers and stronger lung T-cell responses, which are critical for protection against pathogens such as SARS-CoV-2 and malaria.

• Modeling Inflammatory and Antiviral Responses: MIP-3α is an inflammatory cytokine involved in recruiting leukocytes, including lymphocytes and dendritic cells, to sites of injury or infection. It plays a role in innate immunity and has demonstrated antiviral activity in mouse models, making it valuable for studying host-pathogen interactions and immune defense mechanisms.

• Chemotaxis Assays: Recombinant Mouse MIP-3α is used to induce chemotaxis in cell-based assays, particularly with THP-1 cells and BaF3 pro-B cells transfected with CCR6, allowing quantification of chemokine activity and receptor function.

• Immunotherapy Research: Combined administration of MIP-3α with other chemokines (e.g., MIP-1α) can efficiently mobilize dendritic cells, which is beneficial for developing immunotherapeutic strategies and collecting DCs for ex vivo manipulation.

Best practices:

• Use recombinant MIP-3α in controlled chemotaxis assays to validate its activity and specificity for CCR6-expressing cells.
• Store lyophilized MIP-3α desiccated below −18°C and reconstituted protein at 4°C for optimal stability.
• When designing fusion proteins or vaccine constructs, ensure the MIP-3α sequence is homologous to the host species to avoid unwanted immune responses against the chemokine itself.

Summary:
Recombinant Mouse MIP-3α is a versatile tool for immunology research, particularly in studies involving dendritic cell recruitment, vaccine development, and inflammation modeling. Its ability to direct immune responses and enhance antigen presentation makes it highly valuable for both basic and translational research applications.

Yes, recombinant Mouse MIP-3α (CCL20) can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately known. This is a common practice in quantitative ELISA protocols for cytokines and chemokines.

Key considerations and supporting details:

• Recombinant protein as ELISA standard: Most quantitative ELISA kits for mouse MIP-3α use recombinant MIP-3α as the standard to generate the calibration curve. This allows for the quantification of unknown samples by interpolating their absorbance values against the standard curve generated from known concentrations of the recombinant protein.

• Purity and quantification: The recombinant protein used as a standard should be highly purified (typically >95%) and its concentration should be determined accurately, often by absorbance at 280 nm or amino acid analysis. Impurities or inaccurate quantification can lead to errors in sample measurement.

• Preparation and handling: Standards are usually reconstituted in a suitable buffer (often with carrier protein such as BSA to prevent adsorption) and serially diluted to cover the assay’s dynamic range. Follow best practices to avoid repeated freeze-thaw cycles and ensure stability.

• Compatibility with assay antibodies: The recombinant standard should match the native protein in terms of epitope recognition by the capture and detection antibodies used in your ELISA. Most commercial ELISA kits are validated to detect both recombinant and natural forms of MIP-3α. If you are developing your own assay, confirm that your antibodies recognize the recombinant standard equivalently to the native protein.

• Validation: For rigorous quantification, it is recommended to validate that the standard curve generated with recombinant MIP-3α is parallel to the response obtained with native samples (parallelism test). This ensures that the recombinant protein behaves similarly to the endogenous analyte in your assay matrix.

• Intended use: Ensure the recombinant protein is labeled for use as an ELISA standard. Some recombinant proteins are specifically tested for use in ELISA, while others are only validated for bioassays or functional studies. Use only those that are suitable for quantitative ELISA calibration.

Summary Table: Recombinant Mouse MIP-3α as ELISA Standard

In conclusion: If your recombinant Mouse MIP-3α meets these criteria, it is appropriate to use it as a standard for quantification or calibration in your ELISA assays. Always follow best practices for standard preparation and assay validation to ensure accurate results.

Recombinant Mouse MIP-3α (CCL20) has been validated in published research for several key applications, primarily related to its role as a chemokine in immune cell recruitment and function.

Validated Applications in Published Research:

• Chemotaxis Assays:
  Recombinant Mouse MIP-3α is widely used to assess chemotactic responses, particularly for lymphocytes (including T cells, Th17, Tregs, B cells) and dendritic cell (DC) subsets (such as CD11b⁺CD8α⁻ myeloid DCs, Langerhans cells, and immature monocyte-derived DCs). Its bioactivity is commonly measured by its ability to chemoattract CCR6-expressing cells in a dose-dependent manner.

• Bioassays:
  The protein is validated for use in in vitro bioassays to study cell migration, immune cell activation, and signaling through its receptor CCR6.

• Tumor Immunology and Immunotherapy Models:
  Recombinant Mouse MIP-3α has been used in in vivo mouse models to promote dendritic cell recruitment and maturation within the tumor microenvironment, enhancing antigen presentation and anti-tumor immune responses. For example, it has been applied in liver cancer models to facilitate dendritic cell aggregation and improve immunotherapeutic outcomes.

• Vaccine Development:
  MIP-3α has been fused to antigens (e.g., SARS-CoV-2 RBD) to enhance immune responses in vaccine studies, demonstrating increased antibody production and T-cell responses in mice.

• Antimicrobial Activity Assays:
  While most studies focus on human MIP-3α, the chemokine has also been tested for direct antimicrobial activity against bacteria and fungi, leveraging its cationic, amphipathic structure. This application is more established for the human ortholog but is mechanistically relevant for mouse studies.

Additional Notes:

• ELISA and Immunoassays:
  Recombinant Mouse MIP-3α is used as a standard or control in ELISA and multiplex immunoassays to quantify endogenous MIP-3α levels in biological samples.

• Cell Culture and Functional Studies:
  It is applied in cell culture systems to study the effects of chemokine signaling on immune cell differentiation, migration, and function.

Summary Table of Validated Applications

These applications are supported by both primary research articles and product validation data from multiple sources.

Reconstitution of Recombinant Mouse MIP-3α

Initial Preparation Steps

Begin by centrifuging the vial before opening to ensure all lyophilized material is at the bottom. Allow both the vial and reconstitution buffer to equilibrate to room temperature before proceeding. This step is critical for optimal protein solubility and activity.

Reconstitution Protocol

Reconstitute the lyophilized MIP-3α in sterile, distilled water or an appropriate sterile buffer at a concentration of 100 µg/mL or higher. When suspending the protein, gently pipette the reconstitution solution down the sides of the vial rather than using vigorous mixing methods. Do not vortex the protein during reconstitution, as mechanical force can destabilize the protein structure.

Allow the protein to reconstitute for 15-30 minutes with gentle agitation. If visible flakes remain, continue mixing gently at room temperature for up to 2 hours. Use gentle inversion and flicking motions rather than vigorous pipetting to ensure thorough dissolution without denaturing the protein.

Storage Considerations

Short-term Storage

After reconstitution, store the protein at 2-8°C for up to one week, or up to 60 days if maintained at 2-8°C in appropriate conditions. For experiments requiring frequent access, short-term refrigeration is acceptable.

Long-term Storage

For extended storage beyond one week, maintain the protein at -20°C or -80°C (with -80°C being preferred for maximum stability). Aliquots can be stored at -20°C to -70°C for up to 3 months.

Carrier Protein Addition

When preparing stock solutions, consider adding carrier proteins such as 0.2-1% BSA (bovine serum albumin) or HSA (human serum albumin) to enhance protein stability during storage and handling. This is particularly beneficial for long-term storage and when preparing dilute working solutions.

Preparation for Cell Culture Applications

Once reconstituted at the initial concentration, further dilute the stock solution to appropriate working concentrations using sterile aqueous buffers or cell culture medium as needed for your specific experimental protocol. Prepare fresh aliquots for each experiment to maintain optimal biological activity, particularly when using the protein for functional assays such as chemotaxis studies.