Anti-Mouse CD62L [Clone MEL-14] — Purified in vivo PLATINUM™ Functional Grade

Clone MEL-14 is a rat monoclonal antibody targeting mouse CD62L (L-selectin) and is used in vivo mouse studies primarily for two applications: functional blockade of lymphocyte homing to lymph nodes and as a surface marker for flow cytometric identification of lymphocyte subpopulations.

Key uses in in vivo mouse studies:

• Blocking Lymphocyte Homing: MEL-14 can be administered to mice to block the interaction between lymphocytes and high endothelial venules (HEV) in lymph nodes, thereby inhibiting lymphocyte migration or homing to lymph nodes. Pre-incubation of lymphocytes with MEL-14 or systemic administration in mice completely and specifically blocks binding of lymphocytes to HEV in vitro and their migration to lymph nodes in vivo. This property is often exploited in studies analyzing immune cell trafficking, lymphocyte localization, and immune response dynamics.

• Functional Depletion: By blocking CD62L, MEL-14 can functionally deplete naive T cells from entering lymph nodes, which allows researchers to assess the role of lymphocyte trafficking in immunity or inflammation models.

• Phenotyping by Flow Cytometry: Though much of the cited use is in vitro, MEL-14 is also widely used ex vivo (on lymphocytes from mice, sometimes after in vivo exposure to treatments) for flow cytometric identification of naive (CD62L^hi) versus activated/memory (CD62L^lo) T cell populations. Flow cytometry using MEL-14 facilitates the detailed study of T cell subsets, their activation status, and trafficking capacity.

Important considerations:

• Functional Assays: When using MEL-14 in functional blockade studies, sodium azide should be removed from antibody preparations, as it can be toxic in vivo.
• Specificity: MEL-14 binding is specific for mouse CD62L and does not cross-react with other selectins.
• Expression: CD62L is expressed on most T and B lymphocytes, neutrophils, monocytes, and eosinophils, meaning MEL-14 can affect multiple immune cell types.

Summary Table: MEL-14 Uses in In Vivo Mouse Experiments

In summary, MEL-14 is chiefly used in vivo to block lymphocyte trafficking to lymph nodes, allowing researchers to dissect the role of cellular localization in immune responses, as well as to characterize lymphocyte phenotypes ex vivo after in vivo manipulations.

The correct storage temperature for the sterile packaged clone MEL-14 antibody is 2–8°C (typically a standard refrigerator temperature), stored undiluted and protected from prolonged exposure to light. Do not freeze the antibody.

Additional details:

• This temperature range is specific to MEL-14 antibodies as listed by multiple suppliers and applies to both labeled and unlabeled forms.
• Prolonged exposure to light should be avoided to prevent potential degradation, especially for conjugated antibodies.
• General sterile storage recommendations for other types of sterile supplies (not antibodies) may suggest higher temperatures (up to 18–24°C), but antibody reagents like MEL-14 require 2–8°C as specified by manufacturers.

Commonly used antibodies or proteins frequently reported in the literature alongside MEL-14 (anti-mouse CD62L/L-selectin) include markers of T and B cell subsets and activation, as well as additional adhesion molecules and trafficking markers.

Frequently used antibodies/proteins with MEL-14:

• CD3 (pan-T cell marker)
• CD4, CD8 (T helper and cytotoxic T cell markers)
• CD19, B220 (B cell markers)
• CD44 (activation/memory marker, also involved in cell adhesion/migration)
• CD62P (P-selectin) and CD62E (E-selectin): other selectin family adhesion molecules
• CD11a, CD11b, CD18 (integrins: LFA-1, Mac-1-related): leukocyte adhesion and trafficking
• CD34, GlyCAM-1, PSGL-1: endothelial or ligand molecules relevant to leukocyte trafficking and often studied in functional or co-staining experiments
• CCR7, CXCR4: chemokine receptors important for lymphocyte migration and homing

Context and experimental rationale:

• MEL-14 is routinely used to distinguish naïve T and B lymphocytes (high CD62L) from effector/memory (low CD62L) cells in multi-color flow cytometry panels.
• CD62L is also examined in conjunction with other homing and activation markers (e.g., CD44) to define lymphocyte differentiation and migration status.
• In studies on lymphocyte trafficking or tissue residency, MEL-14 may be paired with antibodies to molecules like CD34, GlyCAM-1, or integrins, to study endothelial interactions and the multi-step cascade of lymphocyte migration.

Experimental protocols and applications:

• Flow cytometry panels often include MEL-14 with CD3, CD4, CD8, B220, CD44, and CD11a/CD18 for immunophenotyping.
• Blocking/neutralization experiments targeting CD62L (MEL-14) may use paired controls or isotypes, and sometimes co-blockade with anti-CD44 or anti-integrin antibodies, especially in vivo studies of lymphocyte migration or trafficking.
• For immunoprecipitation or Western blot, additional proteins such as E-selectin, P-selectin, or chemokine receptors may also be included, depending on the migration axis under investigation.

Alternative nomenclature: In the literature, CD62L may also be referenced as L-selectin, LECAM-1, Ly-22, LAM-1, or lymphocyte homing receptor.

In sum, MEL-14 is most often used together with markers for lymphocyte subset identification, activation (e.g., CD44), and other trafficking-related proteins (e.g., integrins, selectins, chemokine receptors) when studying lymphocyte biology and homing in mice.

The MEL-14 clone has generated significant findings in scientific literature, particularly regarding its role in understanding lymphocyte trafficking and selectin function. This rat anti-mouse CD62L monoclonal antibody has been instrumental in advancing our knowledge of cell adhesion mechanisms and immune cell migration.

Recognition and Binding Properties

MEL-14 recognizes mouse L-selectin (CD62L), an adhesion molecule expressed on most T and B lymphocytes, neutrophils, monocytes, and eosinophils. The antibody specifically binds to an epitope within the lectin domain of the murine peripheral lymph node homing receptor (pln HR). Through detailed epitope mapping studies, researchers determined that the MEL-14 recognition site is located within the NH2-terminal 53 amino acids of the lectin domain.

Functional Blocking Capabilities

One of the most significant findings is MEL-14's ability to completely and specifically block lymphocyte-endothelial interactions. Pre-incubation of lymphocytes with MEL-14 completely blocks binding of lymphocytes to high endothelial venules (HEV) in vitro and prevents the migration of lymphocytes to lymph nodes in vivo. This blocking activity demonstrates that MEL-14 interferes with the same cellular interactions that are inhibited by PPME, a yeast cell wall polyphosphomannan carbohydrate.

Structural Insights and Domain Dependencies

Research using MEL-14 revealed important structural requirements for proper epitope recognition. While the antibody recognized truncated homing receptor constructs containing both the lectin and EGF domains, antibody recognition was lost when the lectin domain alone was expressed. This finding suggests that the MEL-14 epitope's conformation may be dependent upon the presence of the EGF domain, highlighting the importance of proper protein folding and domain interactions for antibody recognition.

Mechanistic Understanding of Lymphocyte Trafficking

The functional blocking capabilities of MEL-14 have provided crucial evidence supporting the hypothesis that the peripheral lymph node homing receptor's lectin domain is directly involved in binding lymphocytes to carbohydrate ligands on peripheral postcapillary venule endothelium. This finding has been fundamental to understanding the molecular mechanisms underlying lymphocyte homing and trafficking between blood circulation and lymphoid tissues.

Technical Applications and Research Impact

MEL-14 has proven valuable across multiple research applications, including flow cytometry, immunohistochemistry, and functional assays. Its ability to efficiently block lymphocyte-endothelial interactions has made it an essential tool for studying immune cell migration patterns and understanding the role of selectins in inflammatory processes. The antibody's specificity and blocking function have established it as a standard reagent for investigating L-selectin-mediated cellular interactions in both basic research and applied studies of immune system function.