Anti-Human CXCR4 (Clone 12G5) – Purified in vivo GOLD™ Functional Grade

Clone 12G5 is a monoclonal antibody that specifically recognizes human CXCR4 (CD184) and is extensively used in in vivo mouse studies, particularly involving human tumor xenografts. Its most common applications are:

• Imaging and Detection: 12G5 is used as a specific probe in vivo to map and visualize human CXCR4 expression and distribution in mouse models bearing human tumor xenografts, commonly by radiolabeling for molecular imaging techniques such as PET or SPECT.
• Functional Blocking: It is used to block the interaction between CXCR4 and its ligand CXCL12 (SDF-1), helping to assess the role of this pathway in cell migration, tumor growth, and metastasis within human xenograft models in mice.
• Flow Cytometry, Immunofluorescence, and IHC: Although primarily used in vitro for cell sorting and identification, it is applied in vivo and on ex vivo mouse tissues (containing human cells) for immunohistochemistry (IHC) and immunofluorescence to detect the presence and distribution of human CXCR4+ cells.

Key notes regarding 12G5 and mouse models:

• Species Specificity: 12G5 does not cross-react with mouse CXCR4. Its use in mice is limited to situations where human CXCR4 is expressed, such as in human cell xenograft models or transgenic mice expressing human CXCR4.
• Functional Studies: In vivo, it is used to interrogate the role of human CXCR4 in tumor spread, metastasis, immune cell trafficking, and stem cell engraftment inside mice.
• Co-receptor Blocking (HIV research): Although more common in vitro, 12G5 has also been used to block HIV-1/2 envelope protein (gp120/160) binding to CXCR4 in vivo in specialized preclinical HIV models.

Summary of Main Applications in Mice:

• Tracking human CXCR4+ tumor cell localization and metastasis in xenograft models via imaging.
• Functionally blocking the human CXCR4 pathway to study its impact on tumor biology and immune cell migration.
• Immunohistochemical identification of human CXCR4+ cells in mouse tissues following in vivo experiments.
• Used as a competitive agent for receptor occupancy or blocking studies, e.g., to show specificity of other CXCR4-targeting drugs or probes.

Limitations:
12G5 is not used to interrogate native mouse CXCR4 biology, making its applications niche to humanized or xenograft mouse models.

For all in vivo applications, endotoxin-low preparations are preferred to minimize off-target immune activation in mice.

The 12G5 antibody is commonly used to detect human CXCR4 (CD184) in flow cytometry and related applications. In the literature, it is frequently used alongside specific markers or antibodies to characterize cell populations or study signaling and migration. Common co-used antibodies or proteins include:

• CD4: Given the role of CXCR4 as an HIV co-receptor, studies often simultaneously stain for CD4 to examine T-cell subsets and HIV entry mechanisms.
• Other chemokine receptors: Such as CCR5, which, along with CXCR4, is involved in HIV tropism and immune cell migration.
• Lineage markers (e.g., CD3, CD19, CD45): Used to define T cells, B cells, leukocytes, or hematopoietic populations, especially in immunology and stem cell research.
• Secondary antibodies: Labeled with various fluorophores (e.g., PE, FITC, APC, Alexa Fluor dyes) or enzymes (e.g., HRP, alkaline phosphatase) for detection, depending on assay type.
• CXCL12/SDF-1: The natural ligand for CXCR4 is often used in migration assays, signaling studies, or for blocking/competition experiments, sometimes together with 12G5 to assess specificity or function.
• HIV envelope proteins (gp120) or HIV-1/2 viral strains: When assessing viral binding/inhibition in infectivity assays, 12G5 is used with viral proteins or pseudoviruses.

In summary, 12G5 is most often paired with antibodies to CD4, CCR5, classic immune lineage markers (CD3, CD19, CD45), and detection secondary antibodies. In studies of HIV or chemokine signaling, it may be used with recombinant SDF-1/CXCL12 or viral proteins to dissect functional interactions.

Clone 12G5 is a monoclonal antibody targeting human CXCR4 (CD184), a G-protein-coupled receptor involved in cell migration, immune functions, and tumor biology. Key findings from its citations in scientific literature include:

• Specificity and Application: 12G5 binds specifically to CXCR4 on a wide array of human cells, including hematopoietic stem cells, T cells, B cells, monocytes, macrophages, neutrophils, and eosinophils. It is widely used in flow cytometry to analyze CXCR4 expression on cell surfaces, and its specificity has made it the standard for functional and expression studies of CXCR4.

• Tumor Imaging and Biology: Radiolabeled 12G5 ([^125^I]12G5) accumulates specifically in glioblastoma xenografts in mice, indicating high CXCR4 expression in tumor masses, particularly in hypoxic conditions or due to clonal selection of high-expressing cells. The antibody enables highly specific imaging of CXCR4-expressing tumors and can be used to study tumor microenvironment adaptation.

• Inhibition of Tumor Growth: 12G5 has demonstrated inhibitory effects on proliferation of colon cancer cells, suggesting that blocking CXCR4 may disrupt autocrine signaling loops essential for tumor growth.

• HIV Research: Clone 12G5 inhibits CD4-independent infection by some HIV-2 isolates, as CXCR4 acts as a co-receptor for HIV entry, making 12G5 vital in HIV pathogenesis studies and antiviral screening.

• Functional Antagonism: 12G5 is regularly used in competition and inhibition assays to test the effectiveness of CXCR4 antagonists, since antagonists compete with 12G5 for binding to the receptor. This is an established method for characterizing small-molecule and peptide inhibitors of CXCR4.

• Stem Cell Mobilization and Analysis: In clinical stem cell mobilization, 12G5 is employed to stain and quantify CD184/CXCR4 expression on hematopoietic stem and progenitor cells (HSPCs), especially in comparative studies of mobilization agents (e.g., motixafortide with G-CSF).

• Regulation and Conformation Studies: It is also used to analyze regulation of CXCR4 conformation, such as the effects of intracellular signaling molecules (e.g., Rac1) on antibody binding.

• Diagnostic and Disease Associations: CXCR4 detected by 12G5 is implicated in diseases such as lupus, rheumatoid arthritis, multiple sclerosis, and various cancers, due to its key role in cell trafficking, inflammation, and metastasis.

Clone 12G5 is thus recognized as an authoritative tool for CXCR4 identification, imaging, functional analysis, inhibitor screening, and disease research across oncology, immunology, and virology. Its binding characteristics provide both a direct measurement of CXCR4 activity and a competitive agent for testing pharmacological inhibitors.

Dosing regimens of clone 12G5 antibody vary significantly across different mouse models, and there is no universal protocol; regimens are tailored according to species, experimental aims, application (such as flow cytometry or in vivo blocking), and study-specific requirements.

Key context:

• No universal dosing: According to supplier guidelines and summarized expert resources, dosing of clone 12G5 must be customized for the specific mouse model and experimental context. Factors such as mouse strain, disease model, route of administration, expected pharmacokinetics, and desired biological effect all influence dosage selection.
• In vitro use (e.g., FACS): For flow cytometry, clone 12G5 is typically used at concentrations ranging from about 1–10 μg/mL. For example, one protocol reports intracellular staining of cells with clone 12G5 at 10 μg/mL for 1 hour at 4°C.
• In vivo/in vitro functional studies: Published studies using 12G5 as a competitive binding or blocking antibody generally optimize concentration empirically, sometimes referencing the antibody’s dissociation constant (K_d) in the low picomolar range (e.g., K_d ≈ 151.5 pM). This is more relevant for in vitro assays, but informs the amount needed for effective receptor occupancy in mouse plasma.
• Model-specific considerations: Increased proteolytic activity in rodent plasma may lead to faster degradation and thus may require adjusting the dosing or frequency to maintain effective levels. Mouse strain differences (e.g., C57BL/6 vs. DBA/2) can also affect pharmacodynamics and antibody half-life.
• Empirical optimization: Researchers typically start with standard antibody dosing protocols in mice (e.g., 10–30 μg per injection for blocking antibodies), monitor for effective receptor occupancy or blocking activity, and adjust based on pharmacodynamic or pharmacokinetic readouts.

Additional relevant points:

• Database resources: Systematic resources such as ROADMAPS exist to help researchers identify tolerable and effective dosing regimens for antibodies across various mouse strains and tumor models, underscoring the need for model- and application-specific adjustment.
• No standardization in the literature: There is no published consensus on a single dosing protocol for clone 12G5 across models.

In summary, dosing regimens for clone 12G5 are model-specific and require empirical optimization based on the mouse strain, experimental purpose, antibody stability, and desired outcome. Where guidance is needed, consulting reagent datasheets, relevant literature for in vivo or in vitro applications, and pilot dosing studies is the recommended approach.