Recombinant Mouse MIP-3β

Recombinant Mouse MIP-3β (also known as CCL19) is widely used in research applications to study immune cell migration, lymphoid tissue organization, and immune response modulation, particularly due to its role as a potent chemoattractant for T cells, B cells, and dendritic cells.

Key scientific applications and rationale for using recombinant mouse MIP-3β include:

• Chemotaxis Assays: MIP-3β is a strong chemoattractant for T lymphocytes, B lymphocytes, and myeloid progenitor cells. It is commonly used in in vitro migration assays to study the movement and recruitment of these immune cells in response to chemokine gradients.

• Dendritic Cell and T Cell Trafficking: MIP-3β binds to the CCR7 receptor, which is critical for the homing of dendritic cells and naïve T cells to lymph nodes. This makes it essential for experiments investigating lymphocyte trafficking, lymph node architecture, and immune surveillance.

• Immunological Functional Studies: By modulating the migration and positioning of immune cells, recombinant MIP-3β is used to dissect mechanisms of immune activation, tolerance, and lymphoid tissue organization. It is particularly relevant in studies of adaptive immunity, vaccine responses, and tumor immunology.

• Bioassays and Cell Culture: Recombinant MIP-3β is used in bioassays to test cell responsiveness to chemokines, and in cell culture systems to mimic physiological chemokine environments or to drive specific immune cell behaviors.

• Cancer and Infection Models: Because of its role in immune cell recruitment, MIP-3β is used in models of cancer immunology and infectious diseases to study how immune cells are mobilized to sites of infection or tumors, and how this affects disease progression or therapy outcomes.

• Cross-Species Activity: Recombinant mouse MIP-3β can be used in both mouse and, in some cases, human cell systems to study conserved chemokine functions, though species-specific activity should be validated for each application.

Best practices:

• Use recombinant MIP-3β at concentrations optimized for your specific assay, as activity can vary depending on cell type, density, and experimental conditions.
• Confirm the biological activity of each batch using a chemotaxis or functional assay relevant to your research question.

In summary, recombinant mouse MIP-3β is a critical tool for dissecting immune cell migration, lymphoid tissue dynamics, and the orchestration of immune responses in both basic and translational immunology research.

Yes, you can use Recombinant Mouse MIP-3β as a standard for quantification or calibration in ELISA assays, but with important considerations:

Key Points:

• Recombinant proteins are commonly used as standards in ELISA assays because they are highly purified and their concentration can be accurately determined (e.g., by UV spectroscopy, HPLC, or amino acid analysis).
• For accurate quantification, the recombinant standard should be calibrated against a reference standard (if available) and reconstituted and diluted according to the manufacturer’s instructions using the recommended diluent.
• The standard curve must be generated in each assay using serial dilutions of the recombinant protein, and the samples should be assayed under the same conditions.
• Cross-reactivity and specificity should be confirmed: ensure that the antibodies used in your ELISA recognize the recombinant MIP-3β in the same way as the endogenous protein.

Recommendations:

• Use carrier-free recombinant MIP-3β if possible, as carrier proteins (like BSA) can interfere with ELISA measurements.
• Follow the guidelines for preparing ELISA standards (e.g., proper reconstitution, use of appropriate diluent, avoiding repeated freeze-thaw cycles).
• Validate the standard by checking the linearity of dilution and parallelism between the standard curve and your samples.

References:

• Guidelines for Preparing ELISA Standards | Boster Bio Blog
• Recombinant Mouse CCL19 MIP-3beta carrier-free - BioLegend
• The ELISA Standard Save: Calculation of sample concentrations in ...

In summary, recombinant Mouse MIP-3β is suitable for use as an ELISA standard, provided it is properly handled and validated for your specific assay conditions.

Recombinant Mouse MIP-3β (CCL19) has been validated primarily for use in bioassays, especially functional cell-based assays such as chemotaxis, immune cell migration, and T/B lymphocyte activation studies in published research.

Key validated applications in published research include:

• Bioassays (Functional Assays):
  The most common application is in in vitro bioassays to study chemotactic responses, particularly the migration of T cells, B cells, dendritic cells, and myeloid progenitors via CCR7 signaling. These assays often measure cell migration toward a gradient of recombinant MIP-3β.

• Chemotaxis Assays:
  Recombinant MIP-3β is used to induce and quantify the directed migration of immune cells, especially in transwell or microfluidic migration assays. For example, it has been used to assess the chemotactic activity of mouse and human lymphocytes.

• Immune Cell Activation and Trafficking Studies:
  It is used to investigate the regulation of T cell and dendritic cell migration, lymphatic transendothelial migration, and immune surveillance mechanisms. Studies have examined its role in T cell activation, dendritic cell function, and B cell trafficking.

• Tumor Immunology and Vaccine Research:
  Recombinant MIP-3β has been applied in studies of dendritic cell-based vaccines, tumor immunotherapy, and immune tolerance induction in murine models. It is used to enhance or assess immune responses in these contexts.

• Blocking/Neutralization Assays:
  Some sources indicate use in blocking or neutralization experiments to dissect the role of CCL19/CCR7 signaling in immune processes.

• ELISA and Western Blot (less common):
  While primarily used in functional assays, some recombinant CCL19/MIP-3β proteins have been validated for use as standards or controls in ELISA and Western blot applications. However, these are less frequently reported in the context of published functional studies.

Summary Table of Validated Applications

Note:

• The predominant and best-validated application is in bioassays for chemotaxis and immune cell migration.
• Use in ELISA or Western blot is possible but less common for functional studies, and not all recombinant preparations are validated for these applications—always check the specific validation for your protein lot.
• Published research consistently uses recombinant mouse MIP-3β to probe CCR7-mediated immune cell functions in murine models.

Reconstitution Protocol

Initial Preparation

Begin by centrifuging the vial briefly prior to opening to bring the lyophilized powder to the bottom of the tube. Allow the vial and reconstitution buffer to equilibrate to room temperature before proceeding.

Reconstitution Concentration and Buffer Selection

Reconstitute the lyophilized protein at a concentration of 0.1–1.0 mg/mL. Most protocols recommend reconstituting at 100 µg/mL in sterile phosphate-buffered saline (PBS) containing carrier protein. Alternatively, sterile distilled water or high-purity water (18 MΩ-cm) can be used as the reconstitution medium.

Reconstitution Procedure

Gently pipette the reconstitution buffer down the sides of the vial to suspend the protein. If the protein does not fully dissolve immediately, allow the mixture to incubate for 15–30 minutes with gentle agitation at room temperature. If flakes persist, continue mixing for approximately 2 hours at room temperature.

Buffer Composition for Cell Culture

Carrier Protein Addition

For cell culture applications, further dilutions should be prepared in aqueous buffers supplemented with carrier protein at concentrations of 0.1–1.0% bovine serum albumin (BSA) or human serum albumin. This carrier protein prevents non-specific adsorption of the recombinant protein to container surfaces and helps maintain protein stability during storage and handling.

Storage and Stability

Lyophilized Protein Storage

Store unopened lyophilized vials at 2–8°C in a desiccated state. Short-term storage at room temperature (up to 30 days) or at 4°C (up to 6 months) is permissible.

Reconstituted Protein Storage

Store reconstituted protein solutions in working aliquots at −20°C to −80°C for extended storage. Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these can compromise protein integrity and biological activity. Aliquots should contain at least 10 µL of protein solution with carrier protein present.

Biological Characteristics

The recombinant mouse MIP-3β protein is a 9.2 kDa chemokine containing 83 amino acid residues with four highly conserved cysteine residues characteristic of CC chemokines. This protein binds specifically to the chemokine receptor CCR-7 and demonstrates chemotactic activity for T cells and B cells in bioassay applications.