Recombinant Human CCL23/MPIF-1

Recombinant Human CCL23/MPIF-1 is used in research applications to study its roles in immune cell recruitment, immune regulation, hematopoiesis, and disease pathogenesis, particularly in inflammation, cancer, and hematopoietic disorders.

Key scientific reasons to use recombinant CCL23/MPIF-1 include:

• Chemotactic Activity: CCL23 is a potent chemoattractant for resting T lymphocytes and monocytes, making it valuable for studying immune cell migration and trafficking in vitro and in vivo.
• Immune Regulation: It can induce T-cell exhaustion by upregulating immune checkpoint molecules (e.g., CTLA-4, PD-1, TIGIT, TIM-3, LAG-3) on CD8+ T cells, which is relevant for research on tumor immune evasion, immunotherapy, and T-cell biology.
• Hematopoietic Effects: CCL23 inhibits the proliferation and differentiation of myeloid progenitor cells, affecting granulocyte and macrophage development. This is important for studies on hematopoiesis, bone marrow regulation, and myeloid cell disorders.
• Angiogenesis: It promotes angiogenesis via CCR1 activation on endothelial cells, supporting research into vascular biology and tumor microenvironment.
• Disease Modeling: Elevated CCL23 levels are implicated in the pathogenesis of several diseases, including cancer (e.g., ovarian cancer), inflammatory diseases (e.g., rheumatoid arthritis), and brain injury, making recombinant CCL23 useful for disease mechanism studies and biomarker validation.
• Assay Development: Recombinant CCL23 is essential for ELISA standards, antibody validation, and functional bioassays to quantify or detect CCL23 in biological samples.

Best practices for using recombinant CCL23/MPIF-1:

• Use endotoxin-tested, bioactive recombinant protein for cell-based assays to ensure physiological relevance and avoid confounding immune activation.
• Optimize concentration and incubation time based on the specific cell type and experimental endpoint, as CCL23 effects can be dose- and context-dependent.
• Include appropriate controls (e.g., vehicle, unrelated chemokines, receptor antagonists) to confirm specificity of observed effects.

In summary, recombinant human CCL23/MPIF-1 is a versatile tool for dissecting chemokine-mediated immune processes, modeling disease mechanisms, and developing immunological assays.

You can use recombinant human CCL23/MPIF-1 as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is recognized by the antibodies used in your specific ELISA kit and is of comparable quality and purity to the standard supplied with the kit.

Key considerations and supporting details:

• Many commercial ELISA kits for human CCL23/MPIF-1 are validated to detect both natural and recombinant forms of the protein, and their standard curves are often generated using recombinant CCL23. For example, one kit's manual states: "The Human CCL23 ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human CCL23 proteins".
• The standard curve in these assays is typically prepared by serial dilution of a recombinant CCL23 standard, and sample concentrations are determined by comparison to this curve.
• It is essential that the recombinant CCL23 you use is of high purity, correctly folded, and biologically active, as improper folding or modifications can affect antibody recognition and quantification accuracy.
• Always confirm that your ELISA kit's antibodies are validated to recognize the specific recombinant CCL23 you intend to use. Some kits may be optimized for native protein only, but most commercial kits for CCL23 are compatible with recombinant standards.
• If you are developing a custom ELISA, ensure that your recombinant standard is well-characterized and that its concentration is accurately determined, as this will directly impact the reliability of your quantification.

Best practices:

• Use the same buffer and dilution scheme for your recombinant standard as recommended in the ELISA kit protocol.
• Run a standard curve with each assay to ensure consistency and accuracy.
• If substituting a recombinant standard for the kit-supplied standard, validate that the assay's sensitivity, specificity, and dynamic range are maintained.

In summary, recombinant human CCL23/MPIF-1 is suitable as a standard for ELISA quantification, provided it is compatible with your assay's antibodies and protocol. Always verify compatibility and validate performance when making substitutions.

Recombinant Human CCL23/MPIF-1 has been validated for several key applications in published research, primarily in immunology, cell biology, and cancer studies. The most commonly reported applications include:

• Functional assays: Used to study chemotactic activity, immune cell activation, and signaling pathways, particularly via CCR1 receptor.
• ELISA: Employed as a standard or analyte for quantifying CCL23 levels in biological samples such as serum, plasma, and cell culture supernatants.
• Blocking assays: Utilized to investigate the inhibition of CCL23-mediated effects, such as chemotaxis or receptor activation.
• Western blot: Used for protein detection and validation in cell lysates or tissue extracts.

Supporting details and published research applications:

• Chemotaxis and cell migration: CCL23/MPIF-1 is a potent chemoattractant for monocytes, dendritic cells, osteoclasts, and resting T lymphocytes, and has been used in migration assays to study immune cell recruitment.
• Immune modulation: In vitro studies have shown that recombinant CCL23 induces expression of exhaustion markers (CTLA-4, PD-1, TIGIT, TIM-3, LAG-3) on CD8+ T cells, contributing to immunosuppressive tumor microenvironments.
• Signal transduction: CCL23 has been used to stimulate cells in assays measuring downstream signaling events, such as phosphorylation of kinases (e.g., GSK3β, STAT5A/B, SRC) and GTPγS binding assays for receptor activation.
• Hematopoietic progenitor inhibition: Functional assays have validated CCL23’s ability to suppress colony formation by multipotential hematopoietic progenitor cells.
• Angiogenesis studies: CCL23 has been shown to induce angiogenesis via CCR1 activation on vascular endothelial cells in functional assays.
• ELISA standard: Recombinant CCL23 is routinely used as a standard in ELISA kits for quantifying endogenous CCL23 in biological samples.
• Western blot validation: Used for detection and quantification of CCL23 protein in cell and tissue lysates.

Additional relevant information:

• Cell culture and tissue studies: Carrier-free recombinant CCL23 is recommended for cell or tissue culture experiments, while BSA-containing preparations are preferred for ELISA standards.
• Processing and isoforms: In vitro studies have examined CCL23 processing by matrix metalloproteinases (MMPs), identifying stable isoforms for functional analysis.
• Disease models: Recombinant CCL23 has been used in models of ovarian cancer and melanoma to study its role in immune cell exhaustion and tumor progression.

These validated applications are supported by both product validation data and peer-reviewed research, demonstrating the utility of recombinant human CCL23/MPIF-1 in diverse experimental contexts.

To reconstitute and prepare Recombinant Human CCL23/MPIF-1 protein for cell culture experiments, follow these general guidelines based on standard protocols for lyophilized recombinant proteins:

Reconstitution

1. Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitute the protein in sterile PBS or sterile deionized water, unless otherwise specified by the product datasheet. Typical reconstitution concentrations range from 0.1 to 1.0 mg/mL (e.g., 100 µg protein in 100–1000 µL of buffer).
   • For example, reconstituting at 100 µg/mL in sterile PBS is a common starting point.
3. Gently mix the solution by vortexing or pipetting up and down until fully dissolved. Avoid vigorous shaking to prevent protein denaturation.

Preparation for Cell Culture

1. Aliquot the reconstituted protein if not using immediately to minimize freeze-thaw cycles.
2. Store aliquots at –20°C to –70°C for long-term storage. For short-term use (within a week), the reconstituted protein can be kept at 2–8°C under sterile conditions.
3. Thaw aliquots on ice before use and avoid repeated freeze-thaw cycles.
4. Dilute the protein in cell culture medium or buffer to the desired working concentration for your experiment.

Additional Notes

• Always refer to the product datasheet or Certificate of Analysis (COA) for specific instructions, as some proteins may require special buffers or additives.
• For chemotaxis or functional assays, validate the activity of the reconstituted protein using appropriate bioassays.

These steps ensure proper handling and activity of Recombinant Human CCL23/MPIF-1 for cell culture applications.