Recombinant Human MCP-3

Recombinant Human MCP-3 (CCL7) is a versatile chemokine widely used in research to study immune cell recruitment, inflammation, bone biology, and disease mechanisms due to its ability to chemoattract monocytes and regulate macrophage function.

Key scientific applications and rationale for using recombinant human MCP-3 include:

• Immune Cell Recruitment and Inflammation Studies
  MCP-3 is a potent chemoattractant for monocytes, eosinophils, and basophils, acting through CCR1, CCR2, and CCR3 receptors. It is essential for investigating mechanisms of leukocyte trafficking, inflammatory responses, and cytokine signaling in vitro and in vivo.

• Macrophage Function and Tumor Microenvironment
  MCP-3 regulates macrophage activation and protease secretion, influencing tumor invasion and metastasis. Its production by tumor cell lines makes it valuable for studying tumor-stromal interactions and cancer progression.

• Bone Biology and Osteoimmunology
  MCP-3 promotes osteoblast differentiation and bone formation while inhibiting osteoclast differentiation, contributing to increased bone mass and reduced bone loss. Recombinant MCP-3 is used to dissect signaling pathways (e.g., p38 phosphorylation, interferon beta upregulation) and to model bone diseases such as osteoporosis.

• Biomarker and Disease Prognosis Research
  MCP-3 levels serve as biomarkers for disease severity and prognosis in infectious and non-infectious conditions, such as severe fever with thrombocytopenia syndrome (SFTS). Recombinant MCP-3 enables assay development and validation for clinical research.

• Receptor Binding and Signal Transduction Assays
  Recombinant MCP-3 is used in bioassays to characterize chemokine-receptor interactions, screen for agonists/antagonists, and study downstream signaling events.

• Standardization and Reproducibility
  Recombinant proteins offer high purity, batch-to-batch consistency, and defined activity, which are critical for quantitative and mechanistic studies in cell culture, ELISA, and functional assays.

Best practices:

• Use recombinant MCP-3 for controlled stimulation of immune or bone cells in vitro.
• Employ in migration, chemotaxis, and differentiation assays to elucidate MCP-3-dependent pathways.
• Select carrier-free or BSA-containing formulations based on application (e.g., cell culture vs. ELISA standard).
• Store and handle according to recommended protocols to preserve activity.

Summary:
Recombinant human MCP-3 is a powerful tool for dissecting chemokine biology, immune regulation, bone metabolism, and disease mechanisms, offering reproducibility and specificity for diverse research applications.

Yes, recombinant human MCP-3 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and properly validated for your assay system. This is a common practice in quantitative ELISA protocols for cytokines and chemokines such as MCP-3 (CCL7).

Essential context and best practices:

• ELISA kits for human MCP-3 routinely use recombinant MCP-3 as the standard to generate calibration curves for quantification of MCP-3 in biological samples. The standard curve is constructed by serially diluting recombinant MCP-3 and measuring the optical density (OD) at each concentration, which allows interpolation of sample concentrations based on their OD values.

• Validation: It is important to confirm that the recombinant MCP-3 standard is recognized equivalently to native MCP-3 by the antibodies used in your ELISA. Most commercial kits report that their antibodies recognize both recombinant and natural MCP-3, and that standard curves generated with recombinant MCP-3 are parallel to those obtained with native MCP-3 in biological samples.

• Purity and activity: Ensure the recombinant MCP-3 is of high purity, correctly folded, and biologically active. Impurities or misfolded protein can affect quantification accuracy.

• Preparation: Follow the recommended protocol for reconstitution and dilution of the recombinant MCP-3 standard. Prepare fresh dilutions for each assay, as some protocols specify that the standard solution should only be used on the day of preparation.

• Matrix effects: If you are quantifying MCP-3 in complex matrices (e.g., serum, plasma, cell culture supernatant), confirm that the recovery and linearity of the recombinant standard are acceptable in your matrix. Most validated kits report recovery rates between 85–115% and coefficients of variation (CV) below 10%.

• Documentation: Record the lot number, concentration, and preparation details of your recombinant MCP-3 standard for reproducibility and traceability.

Summary of protocol steps for using recombinant MCP-3 as a standard:

• Reconstitute recombinant MCP-3 according to manufacturer or protocol instructions.
• Prepare a serial dilution series covering the expected range of MCP-3 concentrations in your samples.
• Add standard dilutions to the ELISA plate alongside your samples.
• Generate a standard curve by plotting OD versus MCP-3 concentration.
• Quantify sample MCP-3 concentrations by interpolation from the standard curve.

Limitations:

• Do not mix standards or reagents from different manufacturers or sources unless validated for cross-compatibility.
• Use standards within their validity period and avoid repeated freeze-thaw cycles.

In summary: Recombinant human MCP-3 is suitable and widely used as a standard for ELISA quantification, provided it is validated for your assay system and handled according to best laboratory practices.

Recombinant Human MCP-3 (CCL7) has been validated for several key applications in published research, primarily in functional cell-based assays, biomarker studies, and in vivo models.

Validated Applications in Published Research:

• Chemotaxis Assays:
  MCP-3 is widely used to induce chemotaxis (directed cell migration) of monocytes, THP-1 cells, and other leukocytes in vitro, confirming its biological activity as a chemoattractant. For example, recombinant human MCP-3 has been shown to induce chemotaxis of THP-1 cells starting at 10 ng/ml.

• Bioassays for Receptor Activation:
  MCP-3 is used in bioassays to study activation of chemokine receptors (CCR1, CCR2, CCR3) on various cell types, including carcinoma cell lines and immune cells. These assays often measure downstream signaling events or functional responses such as migration or calcium flux.

• Osteoblast and Osteoclast Differentiation Assays:
  In vitro, recombinant human MCP-3 has been validated for promoting osteoblast differentiation and inhibiting osteoclast differentiation in bone marrow-derived cell cultures. This includes measurements of ALP activity, matrix mineralization, and gene expression of osteoblast markers.

• In Vivo Functional Studies:
  Recombinant MCP-3 has been administered to mice to assess its effects on bone remodeling, including increased bone mass, enhanced BMP2-induced bone formation, and partial rescue of RANKL-induced bone loss.

• Biomarker Validation in Clinical Samples:
  MCP-3 levels have been measured in human serum or plasma as a biomarker for disease severity and prognosis, such as in severe fever with thrombocytopenia syndrome (SFTS) and COVID-19. These studies use MCP-3 as a quantitative analyte in ELISA or multiplex cytokine assays.

• Neutrophil Chemotaxis:
  Recombinant MCP-3 has been validated for inducing chemotaxis of primary human neutrophils in functional assays.

Additional Context and Supporting Details:

• MCP-3 is frequently used as a positive control or standard in chemotaxis and receptor-binding assays due to its well-characterized activity.
• Its role as a chemoattractant for monocytes, eosinophils, and other leukocytes is central to studies of inflammation, immune cell migration, and tumor microenvironment.
• MCP-3 is also used in studies investigating its immunoregulatory functions in diseases such as asthma, intestinal inflammation, and cancer.

Summary Table:

These applications are supported by peer-reviewed studies and product validation data, confirming the utility of recombinant human MCP-3 in both basic and translational research.

To reconstitute and prepare Recombinant Human MCP-3 (CCL7) protein for cell culture experiments, follow these best-practice steps based on current protocols and technical recommendations:

1. Preparation and Handling

• Equilibrate the lyophilized vial and your reconstitution buffer (e.g., sterile PBS or sterile distilled water) to room temperature before opening.
• Centrifuge the vial briefly to ensure all lyophilized powder is at the bottom.

2. Reconstitution

• Buffer choice: Most protocols recommend reconstituting MCP-3 at a concentration of 100 μg/mL in sterile PBS (phosphate-buffered saline). If your application is sensitive to animal proteins, use PBS alone; otherwise, you may add 0.1% BSA (bovine serum albumin) as a carrier to enhance stability, especially for long-term storage or low-concentration use.
• Alternative: Some protocols allow reconstitution in sterile distilled water at 0.1–0.5 mg/mL. For cell culture, PBS or a buffer compatible with your assay is preferred.
• Procedure: Add the calculated volume of buffer gently down the side of the vial. Do not vortex or shake vigorously—gently pipette or swirl to dissolve.
• Incubate at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

3. Aliquoting and Storage

• Aliquot the reconstituted protein into small volumes (≥20 μL) to avoid repeated freeze-thaw cycles.
• Short-term storage: Store at 2–8 °C for up to 1 week.
• Long-term storage: Store aliquots at −20 °C to −80 °C. For long-term stability, especially at low concentrations, include a carrier protein such as 0.1% BSA or 5–50% glycerol.
• Avoid repeated freeze-thaw cycles to prevent protein degradation.

4. Working Solution Preparation

• Before adding to cell culture, dilute the stock solution to the desired working concentration using cell culture medium or PBS. If using serum-free medium, avoid BSA or other animal proteins in your diluent.

5. Additional Notes

• Do not vortex or use vigorous pipetting, as this can denature the protein.
• If the protein does not fully dissolve, allow it to mix gently at room temperature for a few hours, or overnight at 4 °C.
• Always consult the specific Certificate of Analysis (CoA) or product datasheet for your batch, as minor formulation differences may exist.

Summary Table: Key Steps for MCP-3 Reconstitution

These steps will help ensure protein stability and biological activity for your cell culture experiments.