Recombinant Mouse L-Selectin

Using Recombinant Mouse L-Selectin in research applications is essential for studying the molecular mechanisms of leukocyte adhesion, migration, and immune cell trafficking, as well as for modeling inflammatory and immune responses in vitro and in vivo.

Key reasons to use recombinant mouse L-Selectin include:

• Modeling Leukocyte-Endothelial Interactions: L-Selectin is a critical adhesion molecule that mediates the initial tethering and rolling of leukocytes on vascular endothelium, a prerequisite for their migration into lymph nodes and sites of inflammation. Recombinant forms allow controlled studies of these interactions in cell-based assays or flow chambers.

• Functional Assays: Immobilized recombinant L-Selectin can induce adhesion of specific cell lines (e.g., U937 cells) in a dose-dependent manner, enabling quantitative analysis of cell adhesion and migration mechanisms.

• Signal Transduction Studies: Engagement of L-Selectin triggers intracellular signaling pathways that regulate integrin activation, chemotaxis, and cell surface receptor expression (e.g., CXCR4), which are central to immune cell function and homeostasis.

• Inflammation and Disease Models: Recombinant L-Selectin is used to dissect its role in leukocyte recruitment during acute and chronic inflammation, autoimmune disease, and even tumor metastasis, as L-Selectin-deficient models show impaired leukocyte trafficking and altered disease outcomes.

• Protein-Protein Interaction and Ligand Binding Studies: The recombinant protein, especially in tagged or Fc-chimera formats, facilitates biochemical assays to identify binding partners, study glycan interactions (e.g., sialyl Lewis X), and map signaling complexes.

• Standardization and Reproducibility: Recombinant proteins provide a consistent, defined reagent for experimental reproducibility, avoiding variability inherent in primary cell or tissue extracts.

• Tool for Antibody Validation and ELISA: Recombinant L-Selectin is commonly used as a standard or positive control in immunoassays, antibody validation, and flow cytometry.

In summary, recombinant mouse L-Selectin is a versatile tool for dissecting the molecular and cellular basis of immune cell trafficking, adhesion, and signaling, and is indispensable for mechanistic studies in immunology, inflammation, and related fields.

Recombinant Mouse L-Selectin can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, properly quantified, and compatible with your assay system. This is a common practice in ELISA development and quantification of target proteins.

Key considerations and supporting details:

• Recombinant proteins are frequently used as ELISA standards because they provide a defined, quantifiable source of the target antigen, allowing for the generation of a standard curve against which unknown samples can be measured.
• Commercial ELISA kits for mouse L-Selectin often use recombinant L-Selectin as the standard included in the kit, demonstrating its suitability for this purpose.
• The recombinant protein should match the native protein’s epitope(s) recognized by the capture and detection antibodies in your ELISA. If the recombinant L-Selectin is truncated, tagged, or otherwise modified, ensure these changes do not interfere with antibody binding or assay performance.
• Carrier-free recombinant protein is preferred for ELISA standards to avoid interference from stabilizing proteins such as BSA, unless your protocol specifically requires a carrier.
• The standard must be accurately quantified (e.g., by BCA, Bradford, or absorbance at 280 nm) and reconstituted or diluted in a buffer compatible with your assay to ensure reliable calibration.
• Validation is essential: If you are developing your own ELISA or substituting the standard in a commercial kit, you should validate the recombinant standard by comparing its standard curve to that of the kit’s original standard or by spike-and-recovery experiments.

Limitations and caveats:

• Not all recombinant proteins are suitable for all ELISA formats. Some recombinant proteins may not be recognized by certain antibody pairs, especially if they lack post-translational modifications or are expressed in non-mammalian systems.
• Some vendors specifically state that their recombinant proteins are not validated for use as ELISA standards in bioassays, so always check the technical datasheet and, if necessary, perform preliminary validation.

In summary:
You can use recombinant Mouse L-Selectin as a standard for ELISA quantification if it is pure, correctly quantified, and recognized by your assay antibodies. Validation against known standards or by recovery experiments is recommended to ensure accuracy.

Recombinant Mouse L-Selectin has been validated in published research primarily for cell adhesion assays, cell-based functional studies, and as a tool for investigating leukocyte migration, T cell homing, and cancer immunotherapy mechanisms.

Key validated applications include:

• Cell Adhesion Assays: Immobilized recombinant mouse L-Selectin has been shown to induce adhesion of U937 cells in a dose-dependent manner, demonstrating its functional activity in vitro for studying selectin-mediated cell adhesion.

• Cell-Based Functional Assays: The recombinant extracellular domain of mouse L-Selectin is used in cell-based assays to investigate selectin-ligand interactions and leukocyte adhesion dynamics.

• Adoptive Cell Therapy and Cancer Immunotherapy Research: Recombinant and genetically modified forms of mouse L-Selectin have been used in mouse models to enhance T cell efficacy in controlling solid and disseminated tumor growth, supporting its role in immunotherapy studies.

• Leukocyte Migration and Homing Studies: L-Selectin is a key molecule for lymphocyte homing to secondary lymphoid organs and migration to sites of inflammation, and recombinant forms are used to probe these processes in vitro and in vivo.

• Marker for Cell Subset Isolation: Recombinant L-Selectin and related reagents are used for positive selection or depletion of T cell subsets from lymphoid tissues, aiding in immunological research and cell sorting protocols.

Additional validated uses in published research include:

• ELISA Standard: Recombinant L-Selectin is recommended as a standard for ELISA-based quantification of selectin or ligand interactions.

• Investigation of Selectin-Ligand Binding: Recombinant protein is used to detect and characterize functional selectin ligands on tissues and cells, including dynamic biochemical tissue analysis.

• Study of Pathological Processes: Research has utilized recombinant L-Selectin to explore its involvement in viral infection, allergy, sepsis, and autoimmune diseases, as well as its shedding and regulation by metalloproteinases.

These applications are supported by published studies and product validation data, confirming the utility of recombinant mouse L-Selectin in diverse immunological, cell biology, and translational research contexts.

To reconstitute and prepare Recombinant Mouse L-Selectin protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS at a concentration of 0.1 mg/mL unless otherwise specified by the product datasheet. This concentration is suitable for most cell-based assays and functional studies.

Step-by-step protocol:

1. Equilibrate materials: Allow the vial of lyophilized protein and the sterile PBS (or recommended buffer) to reach room temperature before opening.
2. Centrifuge vial: Briefly centrifuge the vial to ensure all powder is at the bottom.
3. Add buffer: Add sterile PBS to achieve the desired concentration (e.g., 0.1 mg/mL). For example, add 1 mL PBS to 0.1 mg of protein.
4. Mix gently: Gently agitate or invert the vial to dissolve the protein. If visible particulates remain, continue mixing for up to 2 hours at room temperature.
5. Aliquot: Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles.
6. Storage: Store reconstituted protein at 4–8°C for short-term use (2–7 days), or at <–20°C for longer-term storage (up to 3 months). Avoid repeated freeze-thaw cycles.

Additional notes for cell culture:

• Use carrier-free protein for cell-based assays to avoid interference from stabilizers or carrier proteins.
• Filter-sterilize the solution if sterility is required for cell culture.
• For functional assays, typical working concentrations range from 0.1–0.5 μg/mL for cell adhesion studies.
• Always consult the specific product datasheet for any unique requirements regarding buffer composition, concentration, or additives.

Summary Table:

This protocol ensures optimal solubility and biological activity of recombinant Mouse L-Selectin for cell culture experiments.