Recombinant Human IL-3 Rα

Recombinant Human IL-3 Rα is used in research applications to study and manipulate hematopoietic cell recruitment, differentiation, and survival, as well as to investigate immune regulation and inflammatory responses.

IL-3 and its receptor (IL-3 Rα) are central to hematopoiesis, supporting the proliferation and differentiation of hematopoietic stem and progenitor cells into mature blood cells, including neutrophils, eosinophils, monocytes, and lymphocytes. This makes recombinant IL-3 Rα valuable for:

• Cell culture protocols: Maintaining and expanding hematopoietic stem cells, macrophage precursors, and other lineage-committed progenitors.
• Bioassays: Assessing cell proliferation, survival, and differentiation, particularly in TF-1 human erythroleukemic cells, which are highly responsive to IL-3 stimulation.
• Inflammation and immune response studies: IL-3 Rα is involved in the regulation of inflammation, with effects on cell adhesion, chemotaxis, migration, and cytotoxicity, especially in eosinophils and other myeloid cells.
• Disease modeling and therapeutic screening: Recombinant IL-3 Rα is used to model hematologic diseases (e.g., myelodysplastic syndromes, leukemia) and to screen for drugs or antibodies that modulate IL-3 signaling.
• Standardization and quantification: Serving as a standard in detection and quantification assays for IL-3 and its receptor, and in release assays for engineered IL-3 or blocking antibodies.

Key advantages of using recombinant human IL-3 Rα include:

• Defined, reproducible activity for experimental consistency.
• Ability to dissect IL-3 signaling pathways and receptor interactions in human cell systems.
• Utility in both basic research (e.g., stem cell biology, immunology) and translational studies (e.g., drug development, therapeutic antibody screening).

In summary, recombinant human IL-3 Rα is a critical tool for research into hematopoiesis, immune regulation, inflammation, and therapeutic development, enabling precise control and analysis of IL-3-dependent cellular processes.

Recombinant Human IL-3 Rα (Interleukin-3 Receptor alpha, CD123) is not appropriate as a standard for quantification or calibration in ELISA assays designed to measure IL-3.

Key points:

• ELISA standards must match the analyte: For quantification of IL-3 in ELISA, the standard must be recombinant or purified human IL-3, not its receptor (IL-3 Rα/CD123). The receptor and the cytokine are distinct proteins with different structures and immunological properties.
• Recombinant IL-3 Rα is used for different assays: Recombinant IL-3 Rα is typically used in binding studies, receptor-ligand interaction assays, or as a standard in ELISAs specifically designed to quantify IL-3 Rα/CD123, not IL-3 itself.
• Assay specificity: ELISA kits for IL-3 quantification use anti-IL-3 antibodies and require IL-3 as the standard. Using IL-3 Rα as a standard would not generate a valid standard curve for IL-3 detection, as the antibodies would not recognize the receptor in the same way as the cytokine.

If your goal is to quantify IL-3:

• Use recombinant human IL-3 as your standard.
• Ensure your ELISA kit is validated for IL-3 and that the standard curve is generated with IL-3, not IL-3 Rα.

If your goal is to quantify IL-3 Rα (CD123):

• Use recombinant human IL-3 Rα as the standard, but only in ELISAs specifically designed for IL-3 Rα quantification.

Summary Table:

Using the wrong standard will result in inaccurate quantification and invalid assay results. Always match the standard to the analyte your ELISA is designed to detect.

Recombinant Human IL-3 Rα (CD123) has been validated in published research primarily for applications involving binding assays, functional bioassays, and studies of receptor-ligand interactions, especially in the context of hematopoietic and immune cell biology.

Key validated applications include:

• Binding Activity Assays: Recombinant Human IL-3 Rα is commonly used to assess the binding of IL-3 or anti-CD123 antibodies, which is critical for characterizing antibody specificity, affinity, and blocking potential in both basic research and therapeutic antibody development.

• Bioassays: It is used in cell-based bioassays to evaluate the functional activity of IL-3, including its ability to stimulate proliferation or differentiation of hematopoietic cells expressing CD123.

• Screening and Release Assays: The protein is employed in screening assays for engineered IL-3 variants or antibodies targeting IL-3Rα, as well as in release assays to ensure the potency and specificity of therapeutic candidates.

• Receptor-Ligand Interaction Studies: Recombinant IL-3 Rα is used to study the molecular mechanisms of IL-3 signaling, including receptor dimerization and downstream pathway activation, which are relevant for understanding hematopoietic cell regulation and for drug discovery.

• Flow Cytometry and Immunoassays: While not always explicitly stated in every publication, recombinant IL-3 Rα is often used as a standard or positive control in flow cytometry and ELISA-based assays to detect or quantify CD123 expression or antibody binding.

Context and Supporting Details:

• The use of recombinant IL-3 Rα is particularly important in the development and validation of monoclonal antibodies or antibody-drug conjugates targeting CD123, which is a therapeutic target in hematologic malignancies such as acute myeloid leukemia.
• Functional validation typically involves demonstrating that the recombinant protein binds IL-3 with high affinity and can be recognized by anti-CD123 antibodies in various assay formats.
• Published research also highlights its use in mechanistic studies of immune cell recruitment, differentiation, and signaling, especially in the context of inflammation and cancer immunotherapy.

If you require details on a specific assay type (e.g., ELISA, SPR, cell-based proliferation), please specify, as the above covers the most broadly validated applications in the literature.

To reconstitute and prepare Recombinant Human IL-3 Rα (CD123) protein for cell culture experiments, follow these general best practices, as specific protocols may vary by supplier and formulation. Always consult the Certificate of Analysis (CoA) or product datasheet for your specific lot for optimal results.

General Reconstitution Protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
• Add sterile buffer: Most recombinant IL-3 Rα proteins are lyophilized from PBS (pH 7.4), often with trehalose as a stabilizer. Unless otherwise specified, reconstitute in sterile PBS (pH 7.4) or endotoxin-free water. If the datasheet specifies, use the recommended buffer.
• Concentration: A typical reconstitution concentration is 100 μg/mL. Avoid reconstituting below 100 μg/mL unless the datasheet allows it.
• Dissolve gently: Add the buffer slowly down the side of the vial and gently swirl or invert to dissolve. Do not vortex or pipette vigorously, as this can denature the protein.
• Aliquot immediately: Divide the reconstituted solution into small aliquots to avoid repeated freeze-thaw cycles.
• Storage: Store aliquots at –20°C or –80°C for long-term storage. For short-term use (up to 1 month), 2–8°C is acceptable. Avoid multiple freeze-thaw cycles.

For cell culture applications:

• Dilution: Before adding to cell cultures, dilute the stock solution into your culture medium. It is best practice to include a carrier protein (e.g., 0.1% BSA or HSA) in the dilution buffer to minimize adsorption and stabilize the protein.
• Working concentration: The optimal working concentration depends on your assay but is typically in the range of 0.1–1.0 μg/mL for receptor binding studies or functional assays. For IL-3 itself, bioactivity is often observed at 0.1–1.0 ng/mL, but for the receptor, titrate as needed for your application.

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Critical notes:

• Always check the CoA or datasheet for your specific product, as formulation and recommended buffers may vary.
• If the protein is supplied with trehalose or other excipients, use the recommended buffer to avoid precipitation or loss of activity.
• For functional assays, perform a dose-response to determine the optimal concentration for your cell type and application.

If you provide the exact formulation or supplier, more tailored instructions can be given.