Recombinant Mouse MCP-3

Recombinant Mouse MCP-3 (Monocyte Chemotactic Protein-3, also known as CCL7) is a versatile chemokine widely used in research for its ability to modulate immune cell migration, regulate bone metabolism, and model inflammatory responses. Its use is justified in studies requiring precise control over chemokine concentration and activity, as well as in mechanistic investigations of cell signaling and disease models.

Key reasons to use recombinant mouse MCP-3 in research applications:

• Immune Cell Recruitment and Activation: MCP-3 is a potent chemoattractant for a broad range of leukocytes, including monocytes, T lymphocytes, and other immune cells, by binding to multiple chemokine receptors (e.g., CCR1, CCR2, CCR3, CCR5). This makes it valuable for studying immune cell trafficking, inflammation, and immune response modulation in vitro and in vivo.

• Bone Biology and Osteoimmunology: MCP-3 has been shown to enhance osteoblast differentiation and bone formation while inhibiting osteoclast differentiation, leading to increased bone mass and reduced bone loss in mouse models. It acts via the CCR3 receptor and related signaling pathways, making it a useful tool for investigating bone remodeling, osteoporosis, and bone regeneration.

• Inflammation and Disease Models: MCP-3 plays a critical role in inflammatory responses, such as those seen in infection (e.g., tuberculosis), cancer metastasis, and tissue injury, by regulating monocyte and macrophage recruitment and function. Recombinant MCP-3 enables controlled studies of these processes in cell culture and animal models.

• Chemoattractant Bioassays: Recombinant MCP-3 is commonly used in chemotaxis assays to quantify the migration of specific cell types, such as BaF3-mCCR2 transfectants or primary monocytes, in response to defined chemokine gradients. This is essential for dissecting chemokine-receptor interactions and downstream signaling.

• Defined, Reproducible Experimental Conditions: Using recombinant protein ensures batch-to-batch consistency, defined concentration, and absence of confounding factors present in conditioned media or tissue extracts. This is critical for mechanistic studies and quantitative assays.

• Structure-Function and Receptor Studies: Recombinant MCP-3 allows for mutagenesis, labeling, or modification to probe structure-activity relationships, receptor specificity, and signaling mechanisms in a controlled manner.

Typical applications include:

• In vitro chemotaxis and migration assays
• In vivo models of inflammation, bone remodeling, or immune cell trafficking
• Mechanistic studies of chemokine signaling pathways
• Functional assays for receptor binding and activation

Summary:
Use recombinant mouse MCP-3 when you require a well-characterized, biologically active chemokine to study immune cell migration, bone metabolism, or inflammatory processes in mouse systems, or when you need to dissect chemokine-receptor interactions under defined experimental conditions.

Yes, recombinant Mouse MCP-3 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system. Recombinant MCP-3 is commonly used as a standard in commercial mouse MCP-3 ELISA kits, where it is serially diluted to generate a standard curve for quantifying MCP-3 concentrations in biological samples.

Key considerations and best practices:

• Validation: Ensure the recombinant MCP-3 standard is compatible with the antibodies and detection system used in your ELISA. The standard should produce a dose-dependent signal that is parallel to the signal generated by endogenous MCP-3 in your sample matrix.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant MCP-3 in the same buffer or matrix as your samples to minimize matrix effects. Typical standard curve ranges for mouse MCP-3 ELISA kits are from approximately 8–2000 pg/mL.
• Assay Controls: Include appropriate controls (e.g., blank, low/high standards) to verify assay performance and linearity.
• Recovery and Parallelism: It is recommended to perform spike-and-recovery and parallelism experiments to confirm that the recombinant standard behaves similarly to endogenous MCP-3 in your sample type. This ensures accurate quantification.

Technical protocol example:

• Reconstitute recombinant MCP-3 according to manufacturer instructions.
• Prepare serial dilutions (e.g., 1000, 500, 250, 125, 62.5, 31.25, 15.6 pg/mL) in assay diluent.
• Add standards and samples to the ELISA plate, incubate, and proceed with detection as per your assay protocol.
• Plot the standard curve (OD vs. concentration) and use it to interpolate sample concentrations.

Limitations:

• The recombinant standard must be of high purity and correctly folded to ensure immunoreactivity.
• Quantitative results may differ if the recombinant protein is not identical to the endogenous form in terms of post-translational modifications or folding.

Summary: Recombinant Mouse MCP-3 is suitable as a standard for ELISA quantification, but assay-specific validation is essential for reliable calibration and quantification.

Recombinant Mouse MCP-3 (CCL7) has been validated in published research primarily for applications involving chemotaxis assays, specifically as a chemoattractant for various leukocyte populations, including monocytes, T lymphocytes, and cell lines expressing relevant chemokine receptors.

Key validated applications include:

• Chemotaxis assays: Recombinant mouse MCP-3 has been shown to induce chemotaxis (directed cell migration) of monocytes, T lymphocytes, and THP-1 cells, as well as Baf3 cells transfected with mouse CCR2, in a dose-dependent manner. This is the most widely validated functional application.
• Leukocyte activation studies: MCP-3 is described as a pluripotent chemokine capable of activating multiple leukocyte types, making it useful for studies of immune cell activation and signaling.
• Receptor binding and signaling assays: Its ability to chemoattract cells expressing CCR2 has been used to validate receptor-ligand interactions and downstream signaling events.
• Cell culture supplementation: MCP-3 is used in cell culture to study immune cell migration, differentiation, and cytokine response, although specific protocols may vary.

Additional context:

• The protein is typically validated for in vitro use, including cell migration and activation assays, but not for diagnostic or therapeutic applications.
• Published research also highlights its role in regulating macrophage functions and its potential involvement in tumor biology, such as cancer cell invasion and metastasis, through modulation of macrophage activity.

No evidence was found in the provided search results for validation in applications such as Western blotting, ELISA, or immunohistochemistry for the recombinant protein itself (as opposed to antibodies against MCP-3). The primary focus remains on its bioactivity in functional cell-based assays.

To reconstitute and prepare Recombinant Mouse MCP-3 (CCL7) protein for cell culture experiments, follow these best-practice steps:

1. Centrifuge the vial before opening to ensure all lyophilized protein is at the bottom and to maximize recovery.

2. Reconstitution buffer: Use sterile water or a sterile, physiological buffer such as PBS (phosphate-buffered saline) or 10 mM HEPES, pH 7.4. For cell culture applications, it is common to add a carrier protein (e.g., 0.1–1% BSA or HSA) to minimize protein loss due to adsorption, especially if you plan to store aliquots.

3. Reconstitution concentration:

   • Do not reconstitute to less than 100 μg/mL in water to maintain protein stability.
   • Typical stock concentrations are 50–100 μg/mL, but you may adjust based on your experimental needs.

4. Reconstitution procedure:

   • Add the calculated volume of buffer directly to the vial.
   • Gently pipette up and down and wash down the sides of the vial to ensure complete dissolution.
   • Allow the protein to fully dissolve at room temperature for 10–15 minutes, mixing gently (do not vortex).

5. Aliquot and storage:

   • After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles.
   • Store aliquots at –20°C or –80°C for long-term storage. For short-term use (up to 1 week), store at 4°C.
   • If a carrier protein was not used during reconstitution, add it before freezing for long-term storage to enhance stability.

6. Working solution preparation:

   • Dilute the stock solution to the desired working concentration using cell culture medium or assay buffer immediately before use.
   • Avoid repeated freeze-thaw cycles, as this can reduce protein activity.

Summary of key points:

• Use sterile technique throughout.
• Reconstitute at ≥100 μg/mL in sterile water or buffer, with 0.1–1% BSA/HSA if storing.
• Gently mix and fully dissolve the protein.
• Aliquot and store at –20°C or –80°C; avoid repeated freeze-thaw cycles.
• Prepare working dilutions fresh for each experiment.

These steps will help ensure the integrity and bioactivity of recombinant Mouse MCP-3 for cell culture applications.