Recombinant Human L-Selectin (Leukocyte Selectin)

Recombinant Human L-Selectin (Leukocyte Selectin) is a valuable tool for research focused on leukocyte trafficking, immune cell adhesion, inflammation, and related signaling pathways. It enables precise, reproducible studies of L-selectin’s molecular interactions and biological functions in both physiological and pathological contexts.

Key reasons to use recombinant human L-selectin in research applications:

• Modeling Leukocyte-Endothelial Interactions: L-selectin mediates the initial "rolling" of leukocytes on vascular endothelium, a critical step in leukocyte migration into lymph nodes and inflamed tissues. Recombinant protein allows in vitro reconstitution of these interactions for mechanistic studies and drug screening.

• Studying Inflammation and Immune Cell Recruitment: L-selectin is essential for leukocyte recruitment during acute and chronic inflammation, as well as in autoimmune diseases. Using recombinant L-selectin enables controlled assays to dissect these processes and evaluate potential therapeutic interventions.

• Ligand Binding and Specificity Assays: Recombinant L-selectin can be used in binding assays to identify and characterize its ligands, such as glycosylated adhesion molecules (e.g., GlyCAM-1, CD34, MAdCAM-1, and synthetic glycosulfopeptides). This is crucial for understanding selectin-mediated cell recognition and for developing inhibitors.

• Functional Cell-Based Assays: The protein is used in flow cytometry, bioassays, and cell adhesion assays to study how L-selectin engagement affects leukocyte activation, chemotaxis, and integrin-mediated adhesion. For example, L-selectin stimulation can enhance chemokine-induced adhesion and migration of lymphocytes.

• Cancer and Transplantation Research: L-selectin’s role in lymphocyte homing and tumor immunity is increasingly recognized. Recombinant L-selectin is used to study T-cell trafficking to tumors and to optimize cell therapies (e.g., CAR-T cell engineering). It is also relevant for improving hematopoietic stem cell engraftment in transplantation models.

• Biochemical and Structural Studies: Recombinant protein is essential for structural biology, mutagenesis, and biophysical analyses to elucidate the molecular basis of selectin-ligand interactions.

• Standardization and Reproducibility: Recombinant L-selectin provides a consistent, defined reagent for quantitative and comparative studies, unlike primary cell-derived protein, which can vary in expression and glycosylation.

Common applications include:

• Flow cytometry (as a ligand or blocking reagent)
• Cell adhesion and migration assays
• Ligand binding and inhibition studies
• Western blot and ELISA controls
• Functional studies of immune cell signaling and trafficking

In summary, recombinant human L-selectin is indispensable for dissecting the molecular mechanisms of leukocyte adhesion, migration, and immune regulation, and for developing targeted therapies in inflammation, autoimmunity, cancer, and transplantation.

Yes, recombinant human L-Selectin (Leukocyte Selectin) can generally be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system. Most commercial ELISA kits for human L-Selectin/CD62L are designed to detect both natural (endogenous) and recombinant forms, and their standard curves are often generated using recombinant protein.

Key considerations and supporting details:

• Recognition of Recombinant Protein: Multiple ELISA kits explicitly state that their antibodies recognize both natural and recombinant human L-Selectin, and that recombinant protein is suitable for use as a standard.
• Standard Curve Preparation: The standard curve in these assays is typically generated using serial dilutions of recombinant L-Selectin, allowing for accurate quantification of unknown samples.
• Validation: It is important to ensure that the recombinant protein you use as a standard is of high purity, has a defined concentration, and matches the epitope(s) recognized by the antibodies in your ELISA. Some kits specify the amino acid range or post-translational modifications of the recombinant standard used (e.g., W39-N332).
• Assay Compatibility: Always confirm that your recombinant standard is compatible with the specific ELISA kit or custom assay you are using. Some rare exceptions exist where a kit may not recommend recombinant protein as a standard due to differences in glycosylation or folding, but this is not typical for L-Selectin ELISAs.
• Carrier Proteins: If your recombinant L-Selectin is supplied with a carrier protein (e.g., BSA), ensure this does not interfere with your assay or sample matrix.

Best Practices:

• Use recombinant L-Selectin that is as similar as possible to the native soluble form found in human samples, especially regarding glycosylation and sequence.
• Prepare a fresh standard curve for each assay run, using the same buffer as your samples for dilution.
• If using a custom or in-house ELISA, validate the recombinant standard by comparing its detection curve to that of endogenous L-Selectin from biological samples, if possible.

In summary: Recombinant human L-Selectin is widely accepted and used as a standard for ELISA quantification, but always verify compatibility with your specific assay and validate as needed for rigorous quantification.

Recombinant Human L-Selectin (Leukocyte Selectin) has been validated in published research for a range of applications, primarily in the study of cell adhesion, leukocyte migration, inflammation, and as a tool in various biochemical and cell-based assays.

Key validated applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or positive control to detect L-selectin or its ligands.
• Western Blot: Serves as a positive control for protein detection and quantification.
• Immunoprecipitation: Used to pull down L-selectin or associated complexes from cell lysates.
• Flow Cytometry: Applied to analyze cell surface expression or binding interactions involving L-selectin.
• Bioassays/Binding Assays: Utilized to study cell adhesion, particularly the interaction between L-selectin and its ligands, including the adhesion of tumor cells or leukocytes to endothelial cells.
• Functional Cell Adhesion Assays: Immobilized recombinant L-selectin supports the adhesion of specific cell lines (e.g., LS180 human colorectal adenocarcinoma cells), modeling physiological interactions.
• Chemotaxis and Migration Studies: Used to investigate the role of L-selectin in leukocyte rolling, migration, and extravasation in vitro and in vivo.
• Inhibition/Blocking Studies: Recombinant L-selectin is used to test inhibitors or blocking antibodies targeting leukocyte-endothelial interactions.
• Dynamic Biochemical Tissue Analysis: Applied to detect functional selectin ligands on human tissues, particularly in cancer research.
• Cellular Signaling Studies: Used to study downstream signaling events following L-selectin engagement.

Additional research contexts:

• Cancer biology: Analysis of selectin-ligand interactions in tumor metastasis and circulating tumor cell adhesion.
• Stem cell transplantation: Assessment of selectin-mediated engraftment efficiency in hematopoietic stem cell models.
• Inflammation and autoimmunity: Investigation of L-selectin’s role in leukocyte recruitment during inflammatory and autoimmune responses.

Summary Table of Validated Applications

These applications are widely supported in the literature and by experimental protocols, making recombinant human L-selectin a versatile reagent for immunology, cell biology, and translational research.

To reconstitute and prepare Recombinant Human L-Selectin (Leukocyte Selectin) protein for cell culture experiments, follow these general guidelines based on standard protocols and product specifications:

Reconstitution Steps

1. Centrifuge the Vial: Before opening, briefly centrifuge the vial to ensure the lyophilized powder is at the bottom.

2. Reconstitution Solution: Reconstitute the protein in sterile distilled water or sterile PBS (phosphate-buffered saline), depending on the manufacturer's recommendation. Typical concentrations range from 0.1 to 0.5 mg/mL.

   • For example, if the vial contains 100 µg of protein, add 200–1000 µL of sterile water or PBS to achieve a concentration of 0.1–0.5 mg/mL.

3. Gentle Mixing: Gently swirl or invert the vial to dissolve the protein. Avoid vortexing or vigorous pipetting to prevent denaturation.

4. Add Carrier Protein (Optional): For long-term storage or to stabilize the protein, add a carrier protein such as 0.1% BSA (bovine serum albumin) or 5% HSA (human serum albumin).

5. Aliquot and Store: Aliquot the reconstituted protein into small volumes to minimize freeze-thaw cycles. Store at -20°C to -80°C for long-term storage. Avoid repeated freeze-thaw cycles.

Preparation for Cell Culture Experiments

1. Dilution: Dilute the reconstituted protein to the desired working concentration in cell culture medium or assay buffer.

2. Sterile Filtration: If necessary, filter the solution through a 0.2 µm filter to ensure sterility.

3. Use: Add the protein to the cell culture system according to your experimental design. Typical concentrations used in adhesion assays range from 0.5 to 2.0 µg/mL, but optimize based on your specific assay and cell type.

Additional Tips

• Check Manufacturer’s Instructions: Always refer to the specific product datasheet or certificate of analysis for any unique requirements or recommendations.
• Stability: For short-term use, store the reconstituted protein at 4°C for up to a week. For longer storage, use aliquots at -20°C or -80°C.

By following these steps, you can ensure the proper reconstitution and preparation of Recombinant Human L-Selectin for your cell culture experiments.