Anti-Mouse RANKL (CD254) – Purified in vivo GOLD™ Functional Grade

Clone IK22/5 is most commonly used in mice for in vivo functional blockade of RANKL (CD254), which is critical for studying the roles of RANKL in immune modulation and bone metabolism. Its primary in vivo application is the neutralization of RANKL signaling to assess the impact on osteoclast differentiation, bone resorption, T cell-dendritic cell interactions, and related pathologies.

Key in vivo applications of IK22/5 in mice include:

• RANKL blockade: Used to inhibit RANKL-mediated signaling pathways, particularly to block osteoclast differentiation and function, thus helping model and study diseases such as osteoporosis, bone metastases, and rheumatoid arthritis.
• Study of immune regulation: Applied to dissect the role of RANKL in T cell and dendritic cell interactions, as well as T cell-dependent immune responses in various disease and inflammation models.
• Investigation of tumor immunology: Used experimentally to modulate immune responses, such as enhancing antitumor immunity in breast cancer by modifying the tumor microenvironment through the RANKL pathway.

Additional validated research applications of IK22/5 (some in vivo, some ex vivo/in vitro):

• Flow cytometry to track RANKL-expressing cells in various tissues.
• Immunohistochemistry for localization and quantification of RANKL-positive cells.
• Functional assays to confirm RANKL neutralization ability in vivo and in vitro.

In summary, clone IK22/5 is widely used for in vivo neutralization of RANKL in mice, enabling the study of bone metabolism, immune function, and disease mechanisms where RANKL is implicated.

Commonly used antibodies or proteins co-studied with IK22/5 (anti-mouse RANKL antibody) in the literature include:

• Anti-PD-1 and anti-PD-L1: Extensively used in mouse cancer and immunotherapy models, often to study synergistic or combinatorial blockade with RANKL inhibition.
• Anti-CTLA-4: Occasionally included in studies of immunomodulation or checkpoint blockade with RANKL inhibition.
• Anti-RANK: Used to label RANK-expressing cells alongside RANKL detection to study receptor-ligand biology.
• B220 (RA3-6B2): Used to identify B cells, frequently employed in immunofluorescence panels with anti-RANKL.
• GL7: Marker for activated germinal center B cells (biotinylated form used in combination with IK22/5).
• Thy1.2 (53-2.1): Used to label T cells or specific cell types in tissue sections.
• Bone cell markers: Assays or stains for osteoblast and osteoclast differentiation (such as CFU-F or Von Kossa staining) are common in studies of bone biology with IK22/5.
• Isotype controls: Rat IgG2a isotype control is used alongside IK22/5 for specificity controls in staining.

Other relevant proteins sometimes included in IK22/5-based studies are biotinylated goat anti-rat IgG as a secondary reagent and markers/assays specific for osteoprotegerin, BMP2, or mineralization, depending on the research focus.

In summary, the most frequent co-reagents are other immune checkpoint antibodies (PD-1, PD-L1, CTLA-4), RANK, B cell and T cell markers, and bone differentiation markers, reflecting the broad use of IK22/5 in both immunology and bone biology research.

The key findings from scientific literature citing clone IK22/5—a monoclonal antibody targeting mouse RANKL (CD254/TRANCE)—focus on its role in bone metabolism, immunology, and cancer research:

• IK22/5 is a widely used tool for blocking RANKL: IK22/5 specifically recognizes and neutralizes mouse RANKL, making it a valuable reagent for both in vitro and in vivo studies of RANKL function in osteoclastogenesis, bone remodeling, and immunity.

• Efficient Inhibition of Osteoclast Differentiation and Bone Resorption: Anti-RANKL (IK22/5) effectively blocks RANKL-dependent osteoclast differentiation, as evidenced by reductions in osteoclast-specific markers (TRAP and CTSK) and TRAP staining in cell culture. In animal studies, repeated administration of IK22/5 led to significant osteopetrosis (increased bone mass due to lack of active osteoclasts) without affecting normal growth or tooth eruption.

• Use in Cancer and Immunotherapy Studies: RANKL is implicated in bone metastasis and tumor growth. IK22/5-mediated RANKL blockade suppresses osteoclastogenesis that supports tumor cell colonization in bone, and enhances the efficacy of PD-1/PD-L1 immune checkpoint blockade in murine models of melanoma and prostate cancer. IK22/5 alone, or in bispecific format targeting both RANKL and PD-1, showed anti-metastatic activity in preclinical models.

• Effects on Immune System and Lymphocyte Differentiation: Genetic and antibody-mediated (IK22/5) inhibition of RANKL revealed critical roles in dendritic cell survival and T/B lymphocyte development. RANKL is upregulated upon T cell activation and IK22/5 neutralization highlights RANKL's contribution to immune modulation.

• Experimental Applications: IK22/5 is validated in flow cytometry, western blot, and in vivo experiments to detect or deplete RANKL activity in mouse cells/tissues. It is frequently used for titration studies, functional inhibition assays, and mechanistic studies of cell signaling related to apoptosis, immune responses, and cell survival.

• Mechanistic Insights: RANKL’s downstream signaling includes activation of antiapoptotic kinase AKT/PKB, SRC kinase, and TRAF6, with IK22/5 frequently used to dissect these pathways in cellular models.

Summary Table: Key Literature Findings on Clone IK22/5

Alternative Uses/Findings: Some studies attempt to use IK22/5 as a reference to compare new RANKL inhibitors or combination therapies, and it is included as a "gold standard" for functional antibody assays against RANKL.

These findings establish clone IK22/5 as a foundational antibody for dissecting RANKL biology in murine systems and for preclinical therapeutic evaluation.

Dosing regimens of clone IK22/5 (anti-mouse RANKL antibody) can vary significantly based on the mouse model, the experimental goal (e.g., blocking vs. detecting), and the application format (in vivo vs. flow cytometry vs. western blot):

In vivo (functional blockade) dosing in mice:

• 2.5 mg/kg, single subcutaneous injection: Used in studies evaluating biological effects during mouse growth, with outcomes monitored over several weeks.
• 1 mg/kg, every 3 days for 21 days: Employed in cancer studies, administered intraperitoneally to block RANKL signaling and monitor outcomes over a 21-day period.
• 100–250 µg per mouse, typically by intraperitoneal injection: This range is commonly cited as effective for blocking RANKL signaling in various disease models, although the exact protocol should be titrated for the specific mouse strain and disease context.

In vitro/reagent-based applications:

• Flow cytometry: Suggested dose is ≤ 0.25–1.0 µg per 1 million cells (in 100 µl), with titration recommended depending on cell type and staining protocol.
• Western blot: Suggested concentration is 1–10 µg/ml.

Mouse strain considerations:

• Studies report using wild type FVB/NJ and transgenic lines, with identical anti-RANKL dosing to allow for proper comparison between genotypes.
• Differences in response and pharmacodynamics may arise depending on the mouse genetic background and underlying disease model, making empirical titration important.

Summary Table:

Key points:

• Dosing is frequently titrated in preliminary experiments.
• Optimal dose may depend on both the mouse model (strain, age, disease context) and the desired degree of RANKL inhibition.
• Refer to batch-specific datasheets and published protocols for the most accurate starting points, adjusting as needed for particular experimental setups.

If a specific disease model or mouse strain is in question, refer to primary literature on that model for empirically validated regimens.