Recombinant Mouse CCL1

Recombinant Mouse CCL1 is a valuable tool in research applications focused on immunology, cell migration, inflammation, and tumor biology due to its specific activity as a chemokine that signals through the CCR8 receptor.

Key scientific applications and rationale for use:

• Immune Cell Regulation: CCL1 is crucial for the survival, proliferation, and function of group 2 innate lymphoid cells (ILC2s), which play a central role in type 2 immune responses, including protection against helminth infections and regulation of allergic inflammation. Supplementing cultures with recombinant CCL1 can directly stimulate ILC2 expansion and cytokine production, making it essential for mechanistic studies of immune cell biology.

• Cell Migration and Chemotaxis Assays: Recombinant CCL1 is widely used to study chemotactic responses, especially in cells expressing CCR8, such as monocytes, NK cells, immature B cells, dendritic cells, and certain tumor cells. It induces cytoskeletal rearrangements and migration, which are critical for investigating mechanisms of immune cell trafficking, tumor metastasis, and lymph node entry.

• Tumor Biology and Metastasis: CCL1/CCR8 signaling has been implicated in tumor cell migration to lymphatic endothelial cells and lymph node metastasis. Recombinant CCL1 can be used to model and quantify these processes in vitro, and to test the effects of CCR8 inhibition or neutralization strategies.

• Treg Conversion and Function: CCL1 promotes the conversion of mesenchymal stem cells to FOXP3+CCR8+ regulatory T cells (Tregs), and modulates Treg suppressive function. Recombinant CCL1 is thus useful for studies on immune tolerance, autoimmunity, and regulatory mechanisms.

• Inflammation and Pain Models: CCL1 is involved in the development of neuropathic pain and inflammatory responses. Recombinant protein can be used to dissect signaling pathways and cellular responses in models of pain and inflammation.

• Bioassays and ELISA Standards: Recombinant CCL1 is used as a standard in ELISA assays and as a positive control in chemotaxis bioassays, ensuring reproducibility and quantification in experimental setups.

Best practices:

• Use recombinant CCL1 at experimentally validated concentrations for chemotaxis, proliferation, or cytokine induction assays (e.g., ED50 for chemotaxis in BaF3-CCR8 cells is typically 0.5–2.0 ng/mL).
• Confirm CCR8 expression in target cells to ensure specific responses.
• Include appropriate controls, such as neutralizing antibodies or CCR8 antagonists, to validate specificity of observed effects.

Summary of scientific value:
Recombinant Mouse CCL1 enables precise, reproducible investigation of CCR8-mediated signaling in immune regulation, cell migration, tumor biology, and inflammation. Its use is essential for dissecting chemokine-driven mechanisms and validating therapeutic targets in mouse models and cell-based assays.

Yes, recombinant Mouse CCL1 can be used as a standard for quantification or calibration in ELISA assays, provided the assay is validated for both natural and recombinant forms. Several ELISA kits and protocols specifically mention the use of recombinant Mouse CCL1 as a standard for generating calibration curves and quantifying CCL1 concentrations in biological samples.

Key considerations and supporting details:

• Assay Validation: Many commercial ELISA kits for Mouse CCL1 are designed to detect both natural and recombinant forms of the protein. For example, protocols and product information indicate that recombinant Mouse CCL1/TCA-3 protein is suitable as a standard for ELISA calibration. The assay should be validated to ensure that the recombinant protein behaves similarly to the endogenous protein in terms of antibody recognition and signal generation.

• Standard Preparation: Recombinant Mouse CCL1 is typically reconstituted and serially diluted to generate a standard curve covering the assay’s detection range (e.g., 7.8 pg/mL to 500 pg/mL). The standard curve is then used to interpolate the concentration of CCL1 in unknown samples.

• Specificity: ELISA kits often specify that they can quantify both natural and recombinant Mouse CCL1, with no significant cross-reactivity or interference reported. This ensures accurate quantification when using recombinant protein as a standard.

• Carrier Proteins: Some manufacturers recommend using recombinant CCL1 with carrier proteins (e.g., BSA) for ELISA standards to improve stability and reproducibility. Carrier-free preparations are also available for applications where additives may interfere.

• Limitations: Always confirm that the recombinant protein’s sequence, post-translational modifications, and folding are representative of the native protein in your sample type. Minor differences can affect antibody binding and quantification accuracy. It is best practice to run parallel controls with native protein when possible.

Protocol summary for using recombinant Mouse CCL1 as an ELISA standard:

• Reconstitute recombinant Mouse CCL1 according to the manufacturer’s instructions.
• Prepare serial dilutions to cover the assay’s dynamic range.
• Run the standard curve in parallel with your samples.
• Use the standard curve to calculate sample concentrations by comparing optical density (OD) values.

In summary: Recombinant Mouse CCL1 is widely accepted and used as a standard for ELISA quantification, provided the assay is validated for both forms and the recombinant protein is properly prepared and handled.

Recombinant Mouse CCL1 has been validated for several key applications in published research, primarily in immunology and cell biology:

• Bioassays: Used to assess chemotactic activity, particularly the recruitment of CCR8-expressing T cells, monocytes, and other immune cells. For example, recombinant CCL1 has been shown to chemoattract BaF3 mouse pro-B cells transfected with mouse CCR8, with a reported ED50 of 0.5–2.0 ng/mL.
• Cell Culture: Applied to study cellular responses, differentiation, and signaling in vitro, including the conversion of mesenchymal stem cells to regulatory T cells (Tregs) and modulation of immune cell function.
• ELISA (Standard): Used as a standard protein for quantifying CCL1 levels in biological samples, such as in studies of allergic airway inflammation and immune cell recruitment.
• Functional Assays: Employed to investigate the biological effects of CCL1 on immune cell migration, activation, and cytokine production, including its role in tumor microenvironment and allergic diseases.
• Blocking Assays: Utilized to study the inhibition of CCL1-mediated effects, often in the context of immune regulation and inflammation.
• Western Blot: Used to detect and quantify CCL1 protein in cell lysates and tissue samples.
• Immunohistochemistry and Immunofluorescence: Applied to localize CCL1 expression in tissues, though direct validation for recombinant protein may be less common.
• Flow Cytometry: Used to analyze CCL1-induced changes in cell surface markers and immune cell populations.
• In Vivo Administration: Recombinant CCL1 has been administered to mice to study its effects on immune cell infiltration and disease models, such as allergic inflammation and tumor immunity.

Representative published research applications include:

• T cell migration and recruitment: CCL1 is critical for recruiting CCR8+ T cells to sites of inflammation and tumors, as demonstrated in murine cancer models and studies of allergic airway disease.
• Regulatory T cell (Treg) function: CCL1 promotes Treg conversion and suppressive activity, with antibodies against CCL1 shown to inhibit Treg function.
• Allergic inflammation and asthma: CCL1 is implicated in the recruitment of Th2 cells and the resolution of airway hyperreactivity.
• Tumor immunology: CCL1 enhances the recruitment of effector T cells to the tumor microenvironment, supporting strategies for CAR T cell therapy in solid tumors.
• Cell signaling and differentiation: CCL1 influences stem cell differentiation and immune cell phenotype in vitro.

Summary Table:

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant Mouse CCL1 in diverse experimental settings.

To reconstitute and prepare Recombinant Mouse CCL1 protein for cell culture experiments, follow these best-practice steps based on current protocols and manufacturer recommendations:

1. Reconstitution

• Check the product datasheet for the recommended reconstitution buffer and concentration, as formulations may vary. Most commonly, lyophilized recombinant mouse CCL1 is reconstituted in sterile PBS (phosphate-buffered saline), sometimes with a carrier protein such as 0.1–1% BSA (bovine serum albumin) or HSA (human serum albumin) to stabilize the protein and prevent adsorption to plastic.
• For example:
  • Some protocols recommend reconstituting at 100 μg/mL in sterile PBS.
  • Others suggest 25 μg/mL in sterile PBS containing at least 0.1% BSA or HSA.
• If the datasheet specifies a different buffer (e.g., water or another buffer), follow that instruction.

2. Reconstitution Procedure

• Add the appropriate volume of sterile buffer directly to the vial to achieve the desired concentration.
• Gently mix by swirling or inverting the vial. Avoid vigorous vortexing or pipetting, which can denature the protein or cause foaming.
• Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.
• If necessary, briefly centrifuge the vial to collect the solution at the bottom.

3. Aliquoting and Storage

• Once fully dissolved, aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Store aliquots at –20°C to –70°C for long-term storage. For short-term use (up to 1 month), storage at 2–8°C is acceptable.
• Avoid repeated freeze-thaw cycles.

4. Working Solution Preparation

• Before adding to cell cultures, dilute the stock solution to the desired working concentration using cell culture medium or sterile PBS with carrier protein if needed.
• Filter-sterilize the working solution if sterility is a concern and the buffer is compatible with filtration.

5. Application in Cell Culture

• Add the recombinant CCL1 to your cell culture at the required final concentration, as determined by your experimental design or literature precedent.
• Include appropriate controls (e.g., buffer alone, carrier protein alone) to account for any effects of the vehicle.

Summary Table: Key Steps for Recombinant Mouse CCL1 Reconstitution

Additional Notes

• If the protein is supplied with stabilizers (e.g., trehalose, mannitol), these do not need to be removed before use.
• Always consult the specific product datasheet for any unique instructions or formulation details.

These steps will help ensure the stability and bioactivity of recombinant mouse CCL1 for your cell culture experiments.