Anti-Mouse CD134 (Clone OX-86) – Purified in vivo PLATINUM™ Functional Grade

Clone OX-86 is a widely used monoclonal rat antibody that functions as an agonist of mouse OX40 (CD134), a critical costimulatory receptor expressed on activated CD4 and CD8 T cells. In in vivo mouse studies, OX-86 is administered to stimulate OX40 signaling, thereby influencing T cell responses for various immunological applications.

Key usage and applications in studies:

• Typical dosing: Mice are commonly injected intraperitoneally (i.p.) with OX-86 at doses ranging from 0.2 to 0.25 mg per injection, often for consecutive days (e.g., 3 or 7 days) depending on the experimental design.
• Immune modulation: OX-86 activates OX40, enhancing the proliferation and function of antigen-specific T cells, including both effector and regulatory subsets.
• Experimental effects: In vivo OX-86 treatment has been shown to:
  • Expand Foxp3? regulatory T cells (Tregs) in blood, spleen, and peripheral tissues, but in some models, these expanded Tregs can have impaired suppressive function.
  • Promote memory CD4? T cell expansion, and, with higher or prolonged doses, further increases the memory compartment.
  • Delay tumor growth and enhance anti-tumor immunity in cancer models.
  • Reduce incidence of type 1 diabetes in NOD mice by expanding regulatory T cell populations.

Representative protocol:

• Inject naïve or disease-model mice i.p. with 0.2–0.25 mg OX-86 per mouse for 3–7 consecutive days.
• Assess immune endpoints (such as Treg and memory T cell expansion) by flow cytometry in blood, spleen, and target organs several days post-treatment.

Research cautions:

• Prolonged or high-dose OX-86 administration can destabilize Treg function and induce inflammatory or autoimmune-like pathology.
• OX-86 specifically activates OX40, so effects are tied to OX40-expressing cell populations.

OX-86 is thus a potent tool for manipulating T cell populations and studying T cell costimulation, Treg biology, tumor immunity, and autoimmune disease mechanisms in mice.

Storage Conditions for Sterile Packaged Clone OX-86

Summary Table

Detailed Guidance

• Short-term storage: All sources agree that the sterile packaged clone OX-86 (anti-mouse CD134) should be stored refrigerated at 2–8°C (typically 4°C is specified).
• Long-term storage: For extended storage, aliquot the antibody and store at -20°C (ImmunoTools) or ? -70°C (Leinco). Avoid repeated freeze-thaw cycles to maintain antibody integrity.
• Special handling: Always store in the dark and follow the specific instructions on the product datasheet, as formulations and recommended practices may vary slightly by manufacturer.
• Formulation notes: The antibody is typically supplied in phosphate-buffered saline (PBS), often without added stabilizers or preservatives. Precipitation may occur over time and can be removed by aseptic centrifugation or filtration.

Recommendations

• Upon receipt: Immediately transfer the antibody to the recommended refrigerator (2–8°C).
• For long-term use: Aliquot and freeze at -20°C or lower, avoiding repeated thawing.
• Always consult the specific product datasheet for any unique handling or storage instructions provided by the manufacturer.
• Do not expose to light and avoid freeze-thaw cycles to preserve stability.

For the sterile packaged clone OX-86, refrigerate at 2–8°C for short-term storage and freeze at -20°C or lower (aliquoted) for long-term storage, while strictly avoiding repeated freeze-thaw cycles.

Based on the research literature, several antibodies and proteins are commonly used alongside OX-86 in experimental studies involving the OX-40/CD134 pathway.

Detection and Analysis Antibodies

In ELISA and flow cytometry applications, researchers frequently use anti-PA tag IgG (clone NZ-1) for detecting OX40L fusion proteins, along with peroxidase-conjugated AffiniPure Goat Anti-Rat IgG (H+L) as secondary detection reagents. These are essential for quantifying OX40-OX40L interactions in binding assays.

Cytokine Analysis Panel

When studying T cell responses activated through OX-40 signaling, researchers commonly employ a comprehensive cytokine detection panel alongside OX-86. This includes antibodies for measuring key immune mediators:

Interleukin-2 (IL-2): Anti-IL-2 capture antibody (clone JES6-1A12) paired with biotin-anti-IL-2 detection antibody (clone JES6-5H4)

Interferon-gamma (IFN-?): Anti-IFN-? capture antibody (clone R4-6A2) with biotin-anti-IFN-? detection antibody (clone XMG1.2)

Interleukin-4 (IL-4): Anti-IL-4 capture antibody (clone 11B11) combined with biotin-anti-IL-4 detection antibody (clone BVD6-24G2)

Interleukin-17A (IL-17A): Anti-IL-17A capture antibody (clone TC11-18H10.1) with biotn-anti-IL-17A detection antibody (clone TC11-8H4)

Related Receptor Controls

To ensure specificity in binding studies, researchers often include control antibodies against related TNF receptor superfamily members. These typically include antibodies against 4-1BB, CD27, and GITR to demonstrate that OX40L fusion proteins do not cross-react with these structurally similar receptors.

OX40L Fusion Proteins

Various engineered OX40L proteins are commonly used in conjunction with OX-86 antibody for functional studies, including MBL-OX40L, Fc-MBL-OX40L, Fc-scOX40L, scOX40L-Fc, and MBL-scOX40L. These fusion proteins help researchers understand the functional consequences of OX40 engagement.

Detection Reagents

Standard detection systems used with OX-86 include HRP streptavidin for signal amplification and 3,3',5,5'-tetramethylbenzidine (TMB) substrate for colorimetric detection in ELISA-based assays.

These companion reagents enable comprehensive analysis of OX-40-mediated immune responses, from initial receptor binding to downstream cytokine production and T cell activation patterns.

Key Findings from Clone OX-86 (Anti-Mouse CD134/OX-40) Citations in Scientific Literature

Clone OX-86 (also known as anti-mouse OX-40 or anti-CD134) is a rat IgG1 monoclonal antibody frequently used in immunological research to target murine CD134, a co-stimulatory molecule expressed on activated T cells. Below are the most significant findings from scientific citations involving this clone:

Biological Role and In Vivo Effects

• CD134/OX-40 Enhances T Cell Function: Binding of OX-40 (CD134) by its ligand (OX-40L) or by agonistic antibodies like OX-86 promotes optimal T cell responses, including increased survival, cytokine production (such as IL-2 and IFN-?), and enhanced memory T cell generation after an immune response.
• Critical for Immune Memory: Depletion of CD134 in mouse models leads to reduced numbers of both CD4+ and CD8+ T cells, underscoring its importance in sustaining the T cell compartment after activation.
• Tumor Biology Involvement: Activation of CD134 via antibodies (including OX-86) has been shown to enhance tumor growth in some models, suggesting that CD134 can act as a tumor suppressor. Disruption of CD134 signaling impairs the immune response to tumors, highlighting its complex role in carcinogenesis.
• Broad Cellular Expression: CD134 is expressed not only on activated CD4+ and CD8+ T cells but also on activated regulatory T cells, B cells, NKT cells, NK cells, and neutrophils.

In Vitro and In Vivo Functional Studies

• Costimulatory Activity: In vitro, OX-86 (alongside OX-40L fusion proteins) significantly increases the production of IL-2 and IFN-? from both CD4+ and CD8+ T cells when co-stimulated with anti-CD3, demonstrating its potent costimulatory effect.
• Comparison with OX-40L Fusion Proteins: In vivo, OX-86 acts as a positive control for OX-40 agonism, eliciting significant antigen-specific T cell responses, though some OX-40L fusion proteins (like Fc-scOX40L) can induce even stronger responses.
• Isotype-Specific Effects: As a rat IgG1 (rIgG1), OX-86 primarily interacts with Fc?RIIb and Fc?RIII, giving it a low activatory-to-inhibitory Fc?R binding ratio. This contrasts with mouse IgG2a antibodies, which interact strongly with all activatory Fc? receptors and may mediate effects through cell depletion. The rIgG1 isotype of OX-86 is thus more likely to exert direct agonistic effects via Fc?RIIb-mediated crosslinking.

Experimental Insights

• Requirement for CD4+ T Cells: Studies using OX-86 in MHC Class II knockout mice showed that CD4+ T cells are required for the expansion of antigen-specific CD8+ (OT-I) T cells, as the absence of CD4+ T cells led to a reduction in the resting memory population without affecting the peak primary response.
• Mechanism of Action: The agonistic activity of OX-86 is thought to depend on Fc?R-mediated crosslinking, particularly through Fc?RIIb, which is distinct from the mechanism of depleting antibodies that engage activatory Fc? receptors.

Conclusion

Clone OX-86 is a critical tool for studying the role of CD134/OX-40 in T cell biology, immune memory, and tumor immunology. Its agonistic activity enhances T cell cytokine production and memory formation, while its isotype (rat IgG1) allows for specific mechanistic insights into Fc? receptor-dependent signaling. These findings have broad implications for understanding T cell costimulation and for developing immunotherapeutic strategies targeting the OX-40 pathway.