CD164 Antibody, Biotin conjugated

What is CD164 and why is it significant in research contexts?

CD164, also known as endolyn, MGC-24v, or multi-glycosylated core protein 24, is an 80-100 kDa transmembrane sialomucin protein expressed by multiple cell types including epithelial cells, T and B cells, monocytes, hematopoietic progenitor cells, and activated basophils . The protein functions as both an adhesion molecule and a negative regulator of cell proliferation, particularly in hematopoietic progenitor cells . CD164 has gained significant research interest due to its roles in cell adhesion, migration, and proliferation, as well as its implications in cancer progression . Within the extracellular domain, human CD164 shares approximately 53% sequence identity with mouse and rat CD164, making it an important target for comparative studies across species .

What are the key functional epitopes of CD164 and how do different antibodies target them?

CD164 contains multiple functional epitopes that can be recognized by different monoclonal antibodies. Research has identified at least four key antibody clones (103B2/9E10, 105A5, N6B6, and 67D2) that target distinct regions of CD164 . These epitopes have been classified into three groups based on their sensitivity to enzymatic treatments:

Importantly, these epitopes show differential expression patterns across tissues, suggesting that cell type-specific post-translational modifications may influence CD164 function . When selecting a CD164 antibody, researchers should consider which epitope is most relevant to their specific biological question.

What are the optimal conditions for using biotin-conjugated CD164 antibodies in flow cytometry?

For optimal flow cytometry results with biotin-conjugated CD164 antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Prepare single-cell suspensions at a concentration of 10^5 to 10^8 cells per test in 100 μL of appropriate buffer (PBS with 1-2% BSA is commonly used)

• Antibody titration: Carefully titrate the antibody to determine optimal concentration; start with ≤0.125 μg per test as a general guideline

• Incubation conditions: Incubate cells with the primary biotin-conjugated antibody for 20-30 minutes at 4°C in the dark

• Washing step: Wash cells twice with buffer to remove unbound antibody

• Secondary detection: Incubate with streptavidin-conjugated fluorochrome for 15-20 minutes at 4°C in the dark

• Final wash: Wash cells twice before analysis

It's critical to include appropriate controls, including isotype controls (mouse IgG3 for antibody clone 502021, or mouse IgG1,κ for clone 67D2) , unstained controls, and single-color controls if performing multicolor analysis. For multiparameter analysis, researchers should verify that there is no spectral overlap between fluorochromes, or implement proper compensation.

How can researchers effectively use CD164 antibodies to study hematopoietic progenitor cells?

CD164 is highly expressed on primitive hematopoietic progenitor cells, particularly on the CD34+high, AC133high, CD38low subset, making CD164 antibodies valuable for characterizing these populations . To effectively study hematopoietic progenitor cells using CD164 antibodies:

• Isolate the target population: Use density gradient centrifugation to isolate mononuclear cells from bone marrow, cord blood, or peripheral blood

• Multiparameter approach: Design a panel that includes biotin-conjugated CD164 antibody alongside markers for primitive progenitors (CD34, AC133, CD38)

• Functional assays: After identifying CD164+ populations, assess their functional properties through:

  • Colony-forming assays to evaluate progenitor potential

  • Proliferation assays (e.g., BrdU incorporation) to examine the negative regulatory effects of CD164 engagement

  • Cell adhesion assays to investigate CD164's role as an adhesion receptor

When interpreting results, researchers should consider that CD164 expression decreases as cells differentiate, with mature neutrophils and erythrocytes showing low or negligible levels . Additionally, different CD164 epitopes may be differentially expressed on various hematopoietic cell subsets, so antibody selection is crucial for accurate characterization.

What techniques are available for studying CD164's subcellular localization?

Recent research has revealed that CD164 can localize to both the cytoplasm and nucleus, suggesting diverse cellular functions . To investigate the subcellular localization of CD164:

• Confocal microscopy: Transfect cells with CD164-EGFP fusion constructs and visualize localization patterns via confocal microscopy. This approach has successfully demonstrated nuclear localization of CD164 in ovarian surface epithelial cells

• Subcellular fractionation: Separate nuclear and cytoplasmic fractions using differential centrifugation, followed by immunoblotting with CD164 antibodies to confirm localization patterns

• Immunofluorescence staining: Fix and permeabilize cells, then stain with biotin-conjugated CD164 antibodies followed by streptavidin-conjugated fluorophores. Co-stain with nuclear markers (DAPI) and other cellular compartment markers

When studying CD164's nuclear function, researchers should consider examining its potential role in gene regulation. For example, CD164 has been shown to enhance CXCR4 promoter activity in a dose-dependent manner, suggesting direct nuclear effects on gene transcription . Chromatin immunoprecipitation (ChIP) assays could be employed to further investigate CD164's potential interaction with specific DNA regions.

How does CD164 contribute to cancer progression and metastasis?

CD164 has been implicated in promoting tumorigenesis through several mechanisms that researchers can investigate using biotin-conjugated CD164 antibodies:

• Increased proliferation: CD164 overexpression in human ovarian surface epithelial (hOSE) cells significantly increases proliferation as measured by BrdU incorporation assays . Researchers can use biotin-conjugated CD164 antibodies to sort cells based on CD164 expression levels and compare their proliferative capacities

• Anti-apoptotic effects: CD164 upregulates anti-apoptotic Bcl-2 and downregulates pro-apoptotic Bax, contributing to cancer cell survival . This can be investigated through apoptosis assays in cells with manipulated CD164 expression

• Anchorage-independent growth: CD164 enhances colony formation in soft agar assays, a hallmark of malignant transformation

• SDF-1α/CXCR4 axis activation: CD164 induces SDF-1α production and CXCR4 expression, activating this critical pathway involved in cancer metastasis

Importantly, CD164 has been shown to localize to the nucleus where it can directly enhance CXCR4 promoter activity . Researchers can utilize CD164 antibodies in chromatin immunoprecipitation assays to further characterize this regulatory function. Therapeutic approaches targeting CD164, such as shRNA-mediated knockdown, have shown promise in inhibiting tumor growth in xenograft models, suggesting CD164 as a potential therapeutic target .

What are the most effective strategies for studying CD164-mediated cellular adhesion?

CD164 functions as an adhesion receptor, particularly in hematopoietic cells. To effectively study CD164-mediated adhesion:

• Cell-cell adhesion assays: Use biotin-conjugated CD164 antibodies to block specific epitopes and assess their impact on cell-cell interactions. The 103B2/9E10 and 105A5 antibodies define functional epitopes that mediate adhesion

• Cell-matrix adhesion assays: Evaluate the role of CD164 in adhesion to extracellular matrix components by pre-incubating cells with blocking CD164 antibodies before plating on matrix-coated surfaces

• Flow-based adhesion assays: Employ microfluidic systems to study CD164's role in dynamic cell adhesion under physiological flow conditions

When designing these experiments, researchers should consider that different CD164 epitopes may have distinct functions. The classification of CD164 antibodies into three classes based on their sensitivity to glycosidases can guide selection of the appropriate antibody to target specific glycosylated functional domains . Additionally, post-translational modifications of CD164 vary by cell type, potentially affecting adhesion properties in a context-dependent manner.

How can researchers effectively use CD164 antibodies in combination with other markers for comprehensive phenotyping?

For comprehensive phenotypic characterization, CD164 antibodies can be effectively combined with other markers in multiparameter analysis:

When designing multiparameter panels, researchers should:

• Consider spectral compatibility of fluorochromes

• Titrate each antibody individually before combining them

• Implement proper compensation controls

• Use fluorescence-minus-one (FMO) controls to set accurate gating boundaries

This approach allows for correlation of CD164 expression with functional properties, signaling pathway activation, and cellular fate decisions.

How can researchers address inconsistent staining patterns with biotin-conjugated CD164 antibodies?

Inconsistent staining with biotin-conjugated CD164 antibodies may result from several technical factors that can be systematically addressed:

• Epitope masking: CD164 epitopes may be masked by differential glycosylation patterns. If encountering inconsistent staining, consider:

  • Testing multiple CD164 antibody clones targeting different epitopes (e.g., 105A5 for class I, 103B2/9E10 for class II, N6B6 or 67D2 for class III epitopes)

  • Employing enzymatic treatments (sialidase, O-glycosidase, N-glycanase) before staining to unmask specific epitopes

• Cell preparation issues: Optimize fixation and permeabilization protocols, as excessive fixation can mask epitopes or destroy antigen recognition sites

• Antibody degradation: Ensure proper storage of biotin-conjugated antibodies (4°C in the dark for reconstituted antibodies, -20 to -70°C for long-term storage)

• Endogenous biotin interference: In biotin-rich tissues, consider using a biotin-blocking step before adding biotin-conjugated primary antibodies

• Secondary detection issues: If using streptavidin-conjugated reporters, verify their functionality with positive controls and optimize streptavidin concentration to avoid oversaturation or high background

Detailed troubleshooting records should be maintained, documenting lot numbers, protocols, and results to identify patterns and optimize conditions systematically.

What factors influence CD164 expression levels across different cellular contexts?

CD164 expression varies significantly across cell types and physiological states, which should be considered when interpreting experimental data:

• Developmental stage: CD164 is expressed on CD34+ intra-aortic cell clusters in week 4-5 human embryos and on primitive hematopoietic progenitors from fetal liver, cord blood, and bone marrow

• Differentiation status: Expression is highest on primitive CD34high, AC133high, CD38low cells and decreases progressively with differentiation, with mature peripheral blood neutrophils and erythrocytes showing low or negligible expression

• Malignant transformation: CD164 expression may be upregulated in cancer cells, as observed in ovarian surface epithelial cells upon malignant transformation

• Tissue-specific post-translational modifications: Differential glycosylation of CD164 in various tissues results in epitope masking, leading to apparent differences in expression when assessed with different antibody clones

• Subcellular localization: CD164 can localize to both the cytoplasm and nucleus, potentially affecting detection depending on the permeabilization protocol used

When comparing CD164 expression across experimental conditions, researchers should maintain consistent protocols and consider these biological variables when interpreting differences in expression levels.

How should researchers evaluate CD164 antibody specificity for their particular application?

Rigorous validation of CD164 antibody specificity is essential for generating reliable results:

• Positive and negative control samples: Include cell types known to express high levels of CD164 (e.g., CD34+ hematopoietic progenitors) and negative controls (e.g., mature erythrocytes)

• Knockdown/knockout validation: Confirm specificity by testing the antibody on CD164 knockdown cells (e.g., using shRNA constructs) to demonstrate reduced signal

• Recombinant protein controls: Use recombinant CD164 proteins as positive controls, particularly those expressed in NS0 mouse myeloma cell lines transfected with human CD164 (amino acids Asp24-Leu197)

• Isotype controls: Include appropriate isotype controls (IgG3 for clone 502021 or IgG1,κ for clone 67D2) at the same concentration as the CD164 antibody

• Cross-reactivity testing: If working across species, verify specificity for the target species, noting that human CD164 shares only approximately 53% sequence identity with mouse and rat homologs

Documentation of these validation steps is essential for publication and reproducibility. Additionally, researchers should report the specific clone, lot number, and concentration used in their methods sections.

What are the latest developments in understanding CD164's role in cancer biology beyond hematopoietic malignancies?

Recent research has expanded our understanding of CD164's role in solid tumors, particularly in ovarian cancer:

• Transcriptional regulation: CD164 has been shown to localize to the nucleus where it can directly enhance CXCR4 promoter activity, suggesting a novel role in transcriptional regulation

• SDF-1α/CXCR4 axis modulation: CD164 induces both SDF-1α production and CXCR4 expression, activating this signaling pathway which is critical for cancer cell migration and metastasis

• Anti-apoptotic mechanisms: CD164 overexpression leads to increased Bcl-2 and decreased Bax expression, contributing to cancer cell survival

• In vivo tumorigenesis: CD164 significantly enhances anchorage-independent growth and in vivo tumor formation in xenograft models

• Therapeutic targeting: shRNA-mediated knockdown of CD164 inhibits tumor growth and increases survival time in xenografted mice, suggesting potential therapeutic applications

These findings highlight CD164 as a multifunctional protein that contributes to cancer progression through diverse mechanisms. Future research using biotin-conjugated CD164 antibodies could further elucidate its role in tumor microenvironment interactions, cancer stem cell biology, and metastatic colonization.

How can CD164 antibodies be utilized in high-dimensional single-cell analysis technologies?

Biotin-conjugated CD164 antibodies can be integrated into emerging single-cell technologies:

• Mass cytometry (CyTOF): Biotin-conjugated CD164 antibodies can be detected with streptavidin-conjugated metal isotopes, allowing integration into high-parameter CyTOF panels for comprehensive immunophenotyping

• Single-cell RNA-seq with protein detection: In protocols like CITE-seq or REAP-seq, biotin-conjugated CD164 antibodies can be used alongside oligonucleotide-tagged streptavidin to simultaneously detect CD164 protein expression and transcriptome profiles at single-cell resolution

• Spatial transcriptomics: CD164 antibodies can be incorporated into spatial protein profiling methods to investigate the relationship between CD164 expression and tissue architecture

• Multiplexed imaging: For highly multiplexed imaging technologies (e.g., Imaging Mass Cytometry, CODEX), biotin-conjugated CD164 antibodies can be included to visualize CD164 expression in the spatial context of the tissue microenvironment

These approaches enable researchers to position CD164 within complex cellular networks and signaling pathways, providing a more comprehensive understanding of its biological functions in normal and pathological contexts.

What are the potential therapeutic applications targeting CD164 in human disease?

Based on current research, several therapeutic strategies targeting CD164 show promise:

• RNA interference approaches: shRNA targeting CD164 has demonstrated efficacy in inhibiting tumor growth in ovarian cancer xenograft models, suggesting potential therapeutic applications

• Blocking antibodies: Antibodies targeting specific functional epitopes of CD164 (e.g., 103B2/9E10 and 105A5) could potentially disrupt CD164-mediated adhesion and signaling functions

• Disruption of CD164-CXCR4 axis: Since CD164 promotes tumorigenesis partly through enhancing the SDF-1α/CXCR4 pathway, combination therapies targeting both CD164 and CXCR4 (e.g., with the CXCR4 antagonist AMD3100) might provide synergistic effects

• CAR-T cell therapy: CD164 expression on certain cancer cells could potentially be exploited for chimeric antigen receptor T-cell therapy, similar to approaches using other surface markers

For researchers developing such therapeutic approaches, biotin-conjugated CD164 antibodies serve as valuable tools for target validation, monitoring target engagement, and assessing therapeutic efficacy in preclinical models.