Anti-Mouse CD366 (Tim-3) [Clone RMT3-23] — Purified in vivo PLATINUM™ Functional Grade

Clone RMT3-23 is a rat monoclonal antibody specific for mouse Tim-3 (CD366), used in in vivo mouse studies primarily to block Tim-3 signaling rather than to deplete Tim-3^+^ cells. In published research:

• RMT3-23 is widely employed for functional blockade of Tim-3, a checkpoint receptor expressed on differentiated Th1 and Tc1 cells, certain regulatory T cells (Tregs), and macrophages.
• In vivo experiments typically involve administration of the antibody to mice (e.g., by intraperitoneal injection) aiming to:
  • Inhibit Tim-3 function and thereby assess its immunoregulatory role (for example, in models of autoimmunity, tumor immunity, or infectious disease).
  • Study immune checkpoint blockade, often in cancer models, sometimes in combination with other checkpoint inhibitors.
• Mechanism of action in vivo:
  • RMT3-23 potently blocks binding of Tim-3 to its known ligands (such as galectin-9, phosphatidylserine, and CEACAM1).
  • Unlike some antibodies, RMT3-23 does not deplete Tim-3^+^ cells in vivo; rather, it can down-modulate surface Tim-3 expression under some conditions, but this requires antibody cross-linking and does not result in cell depletion.
• Flow cytometry and immunohistochemistry: RMT3-23 can also be used ex vivo to stain or identify Tim-3-expressing cells, but its principal in vivo application is functional blockade rather than cell depletion or tracking.

References to specific applications:

• In studies of phagocytosis, RMT3-23 was shown to inhibit Tim-3–mediated clearance of apoptotic cells by macrophages in vivo.
• In checkpoint research, blocking Tim-3 with RMT3-23 slows tumor growth and modulates immune cell function in tumor models.

Key points:

• Purpose: Functional Tim-3 blockade (immune modulation), not depletion.
• Administration: Systemic injection in live mice.
• Readouts: Immune activation, inflammatory disease severity, tumor response, phagocytic activity.
• Does not induce cell depletion: Total numbers of Tim-3^+^ cells are largely unaffected by RMT3-23 administration.

If you require a specific protocol or example for dosing and timing, please specify the disease model or study goal.

Sterile packaged clone RMT3-23 (anti-mouse CD366/TIM-3 monoclonal antibody) should be stored at 2–8°C for short-term storage and at –20°C for long-term storage, but not frozen if the supplier specifies “DO NOT FREEZE.”

• For products from Invitrogen/eBioscience, the instructions are: Store at 4°C (dark), DO NOT FREEZE.
• Leinco Technologies specifies: Stable for one week at 2–8°C; for long-term storage, aliquot without diluting and store at –20°C in a manual defrost freezer.
• BioLegend also recommends: Store undiluted between 2°C and 8°C.
• General monoclonal antibody storage guidelines suggest most antibodies are stable at –20°C for long-term or 2–8°C for short-term, but you must follow the manufacturer’s requirements since some formulations must not be frozen (risk of protein denaturation or loss of activity).

Key points:

• Short-term (up to a week): 2–8°C (refrigerated, not frozen).
• Long-term: If the manufacturer allows, aliquot and store at –20°C (protect from repeated freeze-thaw cycles).
• If the supplier says “DO NOT FREEZE” (as with Invitrogen/eBioscience): Always store at 4°C in the dark.

Always check the product datasheet for lot-specific instructions, as formulations or preservatives may vary and affect stability. Avoid frost-free freezers and minimize freeze-thaw cycles to preserve antibody activity.

RMT3-23 is frequently used in combination with several other antibodies and proteins in research settings, particularly in studies focusing on immune checkpoint inhibition and T cell characterization.

Checkpoint Inhibitor Combinations

RMT3-23 has shown remarkable efficacy when combined with anti-PD-1/PD-L1 antibodies in multiple tumor types. This combination approach has been extensively studied in preclinical cancer models, where the dual blockade of Tim-3 and PD-1/PD-L1 pathways demonstrates enhanced anti-tumor immunity compared to single agent treatments.

Other Anti-Tim-3 Antibody Clones

In comparative studies, RMT3-23 is often evaluated alongside other anti-murine Tim-3 antibody clones, including B8.2C12 (a rat IgG1) and 5D12. These different clones vary in their species origin and isotype, with B8.2C12 being a rat IgG1 while RMT3-23 is classified as a rat IgG2a antibody. Each clone has demonstrated monotherapeutic efficacy in different cancer models - RMT3-23 in WT3 sarcoma and MC38 colon carcinoma, while 5D12 has shown efficacy in EL4 lymphoma.

Flow Cytometry Panel Partners

In flow cytometry applications, RMT3-23 is commonly paired with PE-anti-Mouse CD8 antibodies for T cell subset analysis. This combination allows researchers to simultaneously analyze Tim-3 expression on CD8+ T cell populations, which is particularly relevant since Tim-3 is preferentially expressed by differentiated CD4+ Th1 cells and CD8+ Tc1 (cytotoxic) cells.

Cell Activation Reagents

RMT3-23 is frequently used in studies involving ConA (Concanavalin A) stimulation. Researchers typically stimulate splenocytes with ConA for several days before staining with RMT3-23 to analyze Tim-3 expression on activated T cells, as Tim-3 is expressed on activated Th1 and Tc1 lymphocytes.

Ligand Interaction Studies

In functional studies, RMT3-23 is used alongside Tim-3's natural ligands, including galectin-9, phosphatidylserine (PtdSer), and CEACAM1. However, detailed characterization has shown that RMT3-23 weakly blocks CEACAM1 and PtdSer from binding to Tim-3, but not galectin-9, which is important for interpreting experimental results using this antibody.

The versatility of RMT3-23 in these various experimental contexts makes it a valuable research tool for studying Tim-3 function across different immune cell types and disease models, though researchers should be aware of its specific binding and blocking characteristics when designing experiments.

Clone RMT3-23 is a rat IgG2a monoclonal antibody that targets the mouse immunoregulatory receptor Tim-3 (CD366), widely used in immunology and oncology research. The most significant findings from scientific literature regarding this clone include:

• Epitope Recognition: RMT3-23 binds to a unique, non-overlapping epitope on mouse Tim-3, distinct from other widely used anti-Tim-3 clones such as B8.2C12 and 5D12. This allows for combinatorial or sequential use in experimental settings without cross-blocking interference.
• Lack of Cell Depletion: In vivo administration of RMT3-23 does not lead to depletion of Tim-3+ T cells (including regulatory T cells) but can significantly down-modulate Tim-3 surface expression on these cells. This down-regulation is antibody cross-linking dependent and is significant compared to other anti-Tim-3 clones.
• Applications in Cancer Models: RMT3-23 has demonstrated monotherapeutic efficacy in preclinical mouse tumor models (e.g., WT3 sarcoma), implicating Tim-3 blockade as a strategy to modulate anti-tumor immunity.
• Antigen Distribution: Tim-3, the target of RMT3-23, is expressed on a variety of immune cells, including Th1 and Tc1 lymphocytes, CD11b+ macrophages, Tregs, NK cells, and mast cells. This broad distribution underlines the antibody’s utility in studying various facets of immune regulation.
• Role in Immune Regulation: Through its effect on Tim-3 surface modulation, RMT3-23 has been used to investigate Tim-3’s function as an inhibitory receptor implicated in limiting Th1-mediated inflammation and promoting immunological tolerance in autoimmune and cancer models.

Additional notes:

• RMT3-23 is recommended for use in a variety of applications (e.g., tissue staining, flow cytometry, functional studies), especially in murine models.
• Storage and handling recommendations for laboratory use ensure antibody stability and optimal performance.

These points collectively establish clone RMT3-23 as a critical tool for mechanistic and therapeutic studies targeting Tim-3-mediated immune regulation in mouse models.