Recombinant Human Exodus-2 (CCL21)

Recombinant Human Exodus-2 (CCL21) is a valuable tool for research applications due to its well-characterized biological functions and its central role in immune regulation. Here are several key reasons why you should consider using Recombinant Human Exodus-2 (CCL21) in your research:

1. Immune Cell Migration and Homing

• CCL21 signals through the chemokine receptor CCR7, which is expressed on T cells, B cells, and dendritic cells.
• It plays a critical role in the migration and homing of lymphocytes to secondary lymphoid organs such as lymph nodes and spleen, making it essential for studies on immune cell trafficking and lymphoid tissue organization.

2. Tumor Microenvironment and Immunotherapy Research

• CCL21 has been shown to influence the tumor microenvironment by promoting the infiltration of immune cells, particularly CD8+ T cells, and inhibiting the polarization of immunosuppressive neutrophils (N2 polarization).
• High levels of CCL21 are associated with improved responses to immunotherapy in cancers like hepatocellular carcinoma (HCC), making it a relevant molecule for cancer immunology and immunotherapy studies.

3. Chemotaxis Assays

• Recombinant CCL21 is widely used in chemotaxis assays to study the migration of thymocytes, activated T cells, and dendritic cells.
• It is not chemotactic for monocytes or neutrophils, allowing for selective investigation of lymphocyte migration.

4. In Vitro and In Vivo Models

• The protein is suitable for both in vitro cell culture applications and in vivo animal models, enabling researchers to study immune responses, lymphocyte homing, and tumor-immune interactions in controlled experimental settings.

5. Standardization and Reproducibility

• Recombinant CCL21 provides a consistent and reproducible source of the protein, which is crucial for experimental reliability and comparability across studies.

6. Applications in Cell Culture and Transfection

• It can be used to support cell culture systems, particularly those involving immune cells, and can be incorporated into protocols for cell and RNA extraction, analysis, and spectroscopy.

7. Research on Inflammatory and Autoimmune Diseases

• Given its role in immunoregulatory and inflammatory processes, CCL21 is relevant for studies on autoimmune diseases, chronic inflammation, and immune dysregulation.

8. High Purity and Quality

• Commercially available recombinant CCL21 is typically produced with high purity and undergoes rigorous quality control, ensuring reliable performance in research applications.

Summary

Recombinant Human Exodus-2 (CCL21) is a versatile and well-characterized protein that is essential for studies on immune cell migration, tumor immunology, and inflammatory processes. Its use can enhance the depth and reliability of your research in immunology, cancer biology, and related fields.

Yes, recombinant human Exodus-2 (CCL21) can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols for cytokines and chemokines.

Supporting details and best practices:

• Recognition by ELISA kits: Multiple ELISA kits for human CCL21/Exodus-2 explicitly state that they recognize both natural and recombinant forms of the protein, and use recombinant CCL21 as the standard for generating calibration curves. This ensures that the recombinant protein is suitable for quantification purposes in these assays.

• Purity and quantification: For accurate calibration, the recombinant CCL21 should be highly purified, and its concentration should be determined using a reliable method (e.g., absorbance at 280 nm, BCA assay, or HPLC). Impurities or inaccurate quantification can lead to errors in your standard curve.

• Standard curve preparation: Prepare a serial dilution of the recombinant CCL21 to cover the expected range of your assay (commonly from ~10 pg/mL to several ng/mL, depending on the kit and expected sample concentrations). Follow the specific dilution and reconstitution instructions provided by your ELISA kit or protocol, as these may be lot-specific.

• Matrix compatibility: Ensure that the recombinant standard is diluted in the same buffer or matrix as your samples (e.g., assay buffer, serum, or plasma) to minimize matrix effects and improve quantification accuracy.

• Validation: If you are developing a custom ELISA or using a kit for a novel application, validate that your recombinant CCL21 standard produces a linear, reproducible standard curve and that it is detected equivalently to endogenous CCL21 in your sample matrix.

Summary Table: Use of Recombinant CCL21 as ELISA Standard

In summary: Using recombinant human Exodus-2 (CCL21) as a standard is standard practice for ELISA quantification, as long as the protein is pure, accurately quantified, and compatible with your assay system.

Recombinant Human Exodus-2 (CCL21) has been validated for several key applications in published research, primarily related to its role as a chemokine in immune cell migration and tumor immunology.

Validated Applications in Published Research:

• Functional Assays:
  CCL21 is widely used in chemotaxis assays to study the migration of T cells, dendritic cells, and other CCR7-expressing immune cells. Its ability to induce chemotaxis is a hallmark application, often used to assess the functional response of immune cells to chemokine gradients.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Recombinant CCL21 is used as a standard or as a target antigen in ELISA to quantify CCL21 levels in biological samples or to validate antibody specificity.

• Western Blot:
  CCL21 is used as a positive control or to validate antibody specificity in Western blotting, confirming the presence and molecular weight of the protein in samples.

• Blocking Assays:
  CCL21 is employed in blocking or neutralization assays to study the inhibition of chemotactic responses, often using monoclonal antibodies to block CCL21-mediated migration of naïve T cells.

• Immunohistochemistry:
  Recombinant CCL21 is used to validate antibody staining in tissue sections, particularly to study its expression in lymphoid tissues and inflamed endothelium in diseases such as inflammatory bowel disease and autoimmune skin disorders.

• In Vivo Tumor Models and Immunotherapy Research:
  CCL21 has been used in preclinical mouse models to investigate its effects on the tumor microenvironment, particularly in hepatocellular carcinoma (HCC). It has been shown to enhance the efficacy of anti-PD-1 immunotherapy, modulate neutrophil polarization, and promote CD8^+^ T-cell infiltration.

Additional Context:

• Cell Migration and Homing Studies:
  CCL21 is a potent chemoattractant for lymphocytes and dendritic cells, facilitating their migration to secondary lymphoid organs and sites of inflammation.

• Mechanistic Studies:
  It is used to dissect signaling pathways downstream of CCR7 activation, including studies on immune cell trafficking, lymphoid tissue organization, and immune surveillance.

• Disease Models:
  CCL21 has been implicated in autoimmune diseases, cancer immunology, and chronic inflammatory conditions, making it a valuable tool for modeling these diseases in vitro and in vivo.

Summary Table of Validated Applications

These applications are supported by both product validation data and peer-reviewed research, confirming the utility of recombinant human CCL21 in diverse immunological and translational studies.

Reconstitution of Recombinant Human Exodus-2 (CCL21)

Initial Reconstitution

Recombinant human Exodus-2/CCL21 is typically supplied as a lyophilized powder and requires reconstitution before use. The recommended reconstitution concentration is 0.1 mg/mL in sterile water. However, some protocols recommend a minimum concentration of 100 μg/mL (0.1 mg/mL) to maintain protein stability, with the caveat that concentrations below this threshold are not recommended.

To reconstitute the protein:

1. Centrifuge the vial to concentrate the lyophilized powder at the bottom of the tube before opening.
2. Add sterile, distilled water (or high-purity water with 18 MΩ·cm resistivity) to achieve your desired concentration.
3. Gently pipette and wash down the sides of the vial multiple times to ensure complete recovery of the protein into solution.
4. Allow adequate time for complete dissolution, mixing gently rather than vigorously to avoid protein denaturation.

Storage and Stability

Short-term Storage

After reconstitution, store the protein at 4°C for 2-7 days if immediate use is planned.

Long-term Storage

For extended storage, maintain the reconstituted protein at -20°C or below, with -80°C being the recommended temperature. When storing for prolonged periods, consider adding a carrier protein to enhance stability and prevent protein loss due to adsorption to container walls.

Critical Handling Precautions

• Avoid repeated freeze-thaw cycles, as these significantly compromise protein integrity and biological activity.
• If you require multiple aliquots, prepare them during initial reconstitution and store separately to minimize freeze-thaw exposure.

Preparation for Cell Culture Applications

Sample Preparation

Before use in cell culture experiments, filter the reconstituted protein through a sterile filter to remove any particulates or potential contaminants. Note that some protein loss may occur during filtration, so account for this in your calculations.

Dilution and Use

The reconstituted stock solution can be further diluted in appropriate aqueous buffers or cell culture media to achieve your experimental concentrations. Ensure thorough mixing when preparing working dilutions, and use fresh aliquots for each experiment when possible to maintain protein quality.

Quality Considerations

Verify that all reagents and reconstitution materials are sterile and pyrogen-free, particularly when working with sensitive cell culture systems. The protein should be completely dissolved before use, with no visible particulates or cloudiness in the final solution.