ADORA2A Antibody, FITC conjugated

What is ADORA2A and what are its functional characteristics in cellular systems?

ADORA2A (adenosine A2a receptor) is a G protein-coupled receptor that mediates the activity of adenosine, an endogenous nucleoside generated under conditions of hypoxia, ischemia, or inflammation. The receptor's activity is primarily mediated through G proteins that activate adenylyl cyclase. ADORA2A plays critical roles in multiple physiological systems, particularly in modulating dopamine and glutamate signaling in the central nervous system, where it contributes significantly to neural balance .

At the cellular level, ADORA2A has varied localizations that correspond to its diverse functions. Research using immunofluorescence has demonstrated that ADORA2A is predominantly expressed in one side of the cytoplasm in human sclera fibroblasts, which differs from other adenosine receptors such as ADORA1 (nuclear localization) and ADORA2B (weak expression in both cytoplasm and nuclei) . This differential subcellular localization suggests specialized roles for each receptor subtype.

What are the recommended applications for ADORA2A antibodies in experimental settings?

Based on extensive validation data, ADORA2A antibodies are suitable for multiple experimental applications, with varying degrees of optimization required:

For optimal results, each antibody should be titrated in your specific testing system as performance can vary based on tissue/cell type and experimental conditions .

How do ADORA2A antibodies perform across different species in comparative studies?

Research-validated ADORA2A antibodies demonstrate varying cross-reactivity profiles that should be considered when designing comparative studies:

When conducting cross-species studies, preliminary validation of antibody performance in each species is strongly recommended, as epitope conservation can vary significantly .

What are the optimal protocols for detecting ADORA2A expression using flow cytometry with FITC-conjugated antibodies?

For optimal detection of ADORA2A using FITC-conjugated antibodies in flow cytometry, researchers should follow these methodological guidelines:

Live Cell Surface Detection Protocol:

• Harvest cells in exponential growth phase and wash twice with ice-cold PBS containing 1% BSA

• Resuspend cells at 1-5×10^6 cells/ml in binding buffer

• Incubate with Anti-Human Adenosine A2A Receptor (extracellular)-FITC Antibody (2.5 μg per sample) for 30-45 minutes at 4°C in the dark

• Wash twice with binding buffer

• Analyze immediately by flow cytometry using 488 nm excitation with 530/30 nm bandpass filter

Human cell lines THP-1 (monocytic leukemia) and Jurkat (T-cell leukemia) have been successfully used for cell surface detection of ADORA2A . When performing multiparameter analysis, include appropriate compensation controls and isotype controls (rabbit IgG-FITC) to distinguish specific from non-specific binding.

For fixed/permeabilized protocols (detecting total cellular ADORA2A), a dilution range of 1:100-1:400 has been validated for optimal signal-to-noise ratio .

How should researchers approach experimental design when investigating ADORA2A in cancer progression models?

When designing experiments to investigate ADORA2A in cancer progression models, researchers should consider the following methodological approach based on successful studies in colorectal cancer (CRC):

Recommended Experimental Design Framework:

• Expression Analysis Phase:

  • Determine baseline ADORA2A expression across multiple relevant cell lines (e.g., SW620, HCT116, SW480 for CRC studies)

  • Use both qRT-PCR and Western blotting for comprehensive expression profiling

• Functional Assessment Phase:

  • Employ both loss-of-function (siRNA knockdown) and gain-of-function (plasmid overexpression) approaches

  • Assess multiple phenotypes including:

    • Proliferation (CCK8 assay, colony formation)

    • Migration (wound healing assay)

    • Invasion (transwell assay)

    • Apoptosis (flow cytometry with appropriate markers)

• Mechanism Investigation Phase:

  • Examine key signaling pathway components (e.g., PI3K/AKT pathway proteins)

  • Assess apoptosis-related proteins (Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9)

  • Consider pathway inhibitors to confirm mechanistic involvement

Research has shown that ADORA2A knockdown in CRC cells significantly reduces proliferation, migration, and invasion while promoting apoptosis, suggesting it functions as an oncogene in this context. The PI3K/AKT signaling pathway appears to be a key mediator of these effects .

What are the critical considerations when validating ADORA2A antibody specificity for immunohistochemical applications?

Validating ADORA2A antibody specificity for immunohistochemical applications requires a multi-faceted approach to ensure reliable and reproducible results:

Essential Validation Steps:

• Multiple Detection Methods:

  • Compare immunohistochemistry results with other detection methods (Western blot, qRT-PCR)

  • Concordance across methods significantly increases confidence in specificity

• Genetic Controls:

  • Use tissue from ADORA2A knockout models (e.g., Adora2a^f/f; Rosa26^Cre/ERT2 mice after tamoxifen treatment)

  • Compare with wild-type controls (Rosa26^Cre/ERT2 Adora2a WT)

• Tissue/Cell Type Controls:

  • Include known positive controls (brain tissue has high endogenous expression)

  • Include negative controls (tissues with minimal ADORA2A expression)

  • Always run isotype controls to assess non-specific binding

• Antigen Retrieval Optimization:

  • Test multiple antigen retrieval methods: TE buffer pH 9.0 is recommended for many ADORA2A antibodies

  • Alternative: citrate buffer pH 6.0

• Antibody Titration:

  • Test a range of antibody dilutions (1:50-1:500) to determine optimal signal-to-noise ratio

  • Document lot-to-lot variation, particularly for polyclonal antibodies

Research has demonstrated that subcellular localization of ADORA2A can vary by cell type, with distribution patterns ranging from cytoplasmic to membrane-associated. This heterogeneity underscores the importance of rigorous validation .

How does ADORA2A functionality impact cancer progression and what methodologies best elucidate these mechanisms?

ADORA2A has emerged as a significant factor in cancer progression, particularly in colorectal cancer (CRC). Research methodologies have revealed several key mechanisms:

ADORA2A's Impact on Cancer Progression:

• Cell Proliferation and Invasion:

  • ADORA2A knockdown in SW620 and HCT116 CRC cell lines significantly reduces proliferation (measured by CCK8 assay and colony formation)

  • Migration and invasion capabilities are markedly decreased following ADORA2A silencing (assessed via wound healing and transwell assays)

  • Conversely, ADORA2A overexpression in SW480 cells enhances these malignant phenotypes

• Apoptosis Regulation:

  • ADORA2A modulates the expression of key apoptotic proteins:

    • Inhibits pro-apoptotic proteins (Bax, cleaved caspase-3, cleaved caspase-9)

    • Enhances anti-apoptotic protein expression (Bcl-2)

  • Flow cytometry analysis demonstrates significantly increased apoptosis rates following ADORA2A knockdown

• Signaling Pathway Involvement:

  • PI3K/AKT pathway appears to be a primary mediator of ADORA2A's oncogenic effects

  • ADORA2A knockdown decreases phosphorylation of PI3K and AKT without affecting total protein levels

  • This suggests ADORA2A primarily influences pathway activation rather than protein expression

Recommended Methodological Approaches:

• Combined in vitro and in vivo models for comprehensive assessment

• Both gain-of-function and loss-of-function approaches to establish causality

• Integration of transcriptomic and proteomic analyses to identify downstream effectors

• Patient sample correlation to establish clinical relevance

What role does ADORA2A play in endothelial inflammation and vascular pathologies?

ADORA2A has been identified as a critical regulator of endothelial inflammation and vascular pathologies, particularly in cerebral ischemia. Research using genetic and pharmacological approaches has elucidated several key mechanisms:

ADORA2A's Role in Vascular Pathology:

• Endothelial-to-Mesenchymal Transition (EndMT):

  • ADORA2A promotes EndMT, a process where endothelial cells acquire mesenchymal phenotypes

  • TGFβ2 treatment enhances ADORA2A expression alongside EndMT markers (ACTA2, SM22α, COL1, FN)

  • ADORA2A knockdown via siRNA decreases these mesenchymal markers while increasing endothelial marker CDH5

• Blood-Brain Barrier (BBB) Integrity:

  • Mice with endothelial-specific ADORA2A deletion (Adora2a^ΔiVEC) show reduced BBB leakage after ischemic injury

  • This protection correlates with decreased expression of fibrotic markers (ACTA2, SM22a, COL1, FN, TGFβ2)

• Inflammasome Regulation:

  • ADORA2A inactivation inhibits endothelial inflammation by suppressing the NLRP3 inflammasome

  • This results in downregulation of cleaved caspase 1 and IL-1β expression

  • Reduced leukocyte infiltration is observed in ADORA2A-deficient models

Methodological Approaches for Investigation:

Research has shown that specific inactivation of endothelial ADORA2A mitigates ischemic brain injury and improves post-stroke outcomes, suggesting ADORA2A antagonism as a potential therapeutic approach for vascular protection .

How can researchers effectively assess the role of ADORA2A in ocular diseases and retinal pathologies?

Investigating ADORA2A in ocular diseases requires specialized methodological approaches that address the unique challenges of ocular tissue research:

Research Methodologies for ADORA2A in Ocular Pathologies:

• Tissue-Specific Genetic Models:

  • Generate conditional knockout mice with ocular cell-type specificity:

    • Adora2a^f/f; Rosa26^Cre/ERT2 for inducible global deletion

    • Adora2a^f/f; Cdh5^Cre/ERT2 for endothelial-specific deletion

  • Validate tissue-specific deletion using RT-PCR and Western blotting

• Functional Assessment:

  • Spectral domain-optical coherence tomography (SD-OCT) for retinal structure

  • Fluorescein angiography (FA) for blood flow assessment

  • Optomotor response (OMR) for visual acuity measurement

  • Pattern electroretinography (PERG) for photoreceptor function

• Pathology Models:

  • Laser-induced choroidal neovascularization (CNV) for age-related macular degeneration

  • Vldlr^-/- mouse model for spontaneous subretinal fibrosis

  • Combined with ADORA2A manipulation to assess receptor's role

• Histological and Molecular Analysis:

  • RPE/choroidal flat mounts with antibodies against fibrotic markers (COL1, ACTA2)

  • Quantification of leukocyte infiltration and inflammatory markers

  • Assessment of blood-retinal barrier integrity

Research has demonstrated that inactivation of ADORA2A suppresses endothelial-to-mesenchymal transition in choroidal endothelial cells, which may protect against subretinal fibrosis in age-related macular degeneration. This suggests ADORA2A as a potential therapeutic target for preventing vision loss in neovascular age-related macular degeneration .

How can researchers resolve discrepancies in ADORA2A detection across different experimental platforms?

When faced with inconsistent ADORA2A detection results across different platforms, researchers should systematically address several technical factors:

Troubleshooting Discrepancies in ADORA2A Detection:

• Antibody Epitope Considerations:

  • Verify the exact epitope targeted by each antibody

  • ADORA2A antibodies may target different regions:

    • Extracellular epitopes (e.g., amino acids 146-158 of human ADORA2A)

    • C-terminal regions

    • Internal domains

  • Different epitopes may be differentially accessible in various applications

• Sample Preparation Variables:

  • For Western blotting: Ensure complete protein denaturation; ADORA2A is a membrane protein with 7 transmembrane domains

  • For flow cytometry: Compare live cell surface staining vs. fixed/permeabilized protocols

  • For IHC/ICC: Compare different fixation methods and antigen retrieval protocols

• Expression Level Variations:

  • ADORA2A expression varies significantly between tissues and cell lines

    • High in brain tissue, particularly striatum

    • Variable in cancer cell lines (e.g., high in SW620 and HCT116, lower in SW480)

  • Adjust detection sensitivity accordingly

• Post-translational Modifications:

  • ADORA2A undergoes various modifications that can affect antibody binding

  • Observed molecular weight ranges from 40-45 kDa to 45-60 kDa depending on glycosylation status

• Cross-Validation Approach:

  • When possible, use orthogonal methods:

    • Compare protein detection (WB, IHC) with mRNA assessment (qRT-PCR)

    • Use genetic controls (siRNA knockdown, CRISPR knockout) to validate specificity

    • Consider multiple antibodies targeting different epitopes

If discrepancies persist, consider that they may reflect biological reality rather than technical artifacts—ADORA2A may genuinely show different expression patterns or conformational states across different experimental contexts.

What are the optimal approaches for detecting low-abundance ADORA2A in challenging tissue samples?

Detecting low-abundance ADORA2A in challenging tissue samples requires specialized techniques to enhance sensitivity while maintaining specificity:

Strategies for Low-Abundance ADORA2A Detection:

• Signal Amplification Methods:

  • Consider tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Use highly sensitive chemiluminescent substrates for Western blotting

  • For flow cytometry, consider secondary antibodies with brighter fluorophores than FITC (e.g., PE)

• Sample Enrichment Techniques:

  • For membrane proteins like ADORA2A, consider membrane fraction isolation before analysis

  • Use immunoprecipitation to concentrate ADORA2A before Western blotting

  • For cells, consider culture conditions that may upregulate ADORA2A (e.g., TGFβ2 treatment has been shown to enhance ADORA2A expression)

• Optimized Antibody Selection:

  • Choose high-affinity antibodies with demonstrated sensitivity for low-abundance detection

  • Monoclonal antibodies may offer better signal-to-noise ratio in challenging samples

  • Consider recombinant antibodies for superior lot-to-lot consistency

• Protocol Modifications:

  • Extended primary antibody incubation (overnight at 4°C)

  • Optimized blocking to reduce background while preserving specific signal

  • For flow cytometry, increased antibody concentration may be necessary (test concentrations up to 5 μg per sample)

• Validation Controls:

  • Include positive controls with known high ADORA2A expression (e.g., striatal neurons or certain cancer cell lines)

  • Use genetic approaches to confirm specificity:

    • Compare with siRNA knockdown samples

    • Include tissue from Adora2a knockout models when available

Researchers have successfully detected ADORA2A in human choroidal endothelial cells (hCECs) despite relatively low basal expression by optimizing these approaches, particularly by using extended incubation times and sensitive detection systems .

How should researchers interpret and respond to unexpected subcellular localization patterns of ADORA2A?

Unexpected subcellular localization patterns of ADORA2A require careful interpretation and validation strategies:

Interpretation and Validation of Unusual ADORA2A Localization:

• Contextual Assessment:

  • ADORA2A shows documented variability in subcellular localization:

    • Predominantly in one side of the cytoplasm in human sclera fibroblasts

    • Can show granular cytoplasmic staining, possibly in trafficking vesicles

    • May appear in plasma membrane, throughout cytoplasm, or around intracellular membranous structures

  • Consider whether the observed pattern may be biologically relevant rather than artifactual

• Technical Validation:

  • Confirm with multiple antibodies targeting different epitopes

  • Use tagged ADORA2A constructs (GFP, FLAG) as complementary approaches

  • Employ subcellular fractionation followed by Western blotting to biochemically confirm localization

• Biological Significance Assessment:

  • Investigate whether localization correlates with functional state:

    • Internalization may indicate receptor activation/desensitization

    • Nuclear localization could suggest non-canonical signaling roles

    • Cytoplasmic aggregation might indicate protein quality control issues

• Confocal Microscopy Optimization:

  • Use z-stack imaging to fully characterize 3D distribution

  • Employ co-localization studies with organelle markers:

    • Membrane markers (e.g., Na+/K+ ATPase)

    • Endosomal markers (e.g., Rab5, Rab7)

    • ER/Golgi markers for trafficking assessment

• Functional Correlation:

  • Determine if unusual localization correlates with specific cellular functions

  • In cancer cells, altered ADORA2A localization may correlate with proliferative or invasive phenotypes

  • In neurons, distribution may reflect synaptic vs. extrasynaptic signaling roles

Research has shown that in paraformaldehyde-fixed HepG2 cells, ADORA2A antibodies may reveal granular cytoplasmic staining, possibly representing protein in trafficking vesicles rather than the expected membrane localization. This pattern was reproducible and distinct from non-specific background staining shown with isotype controls .

How can ADORA2A antibodies be effectively employed in understanding immunomodulatory pathways in cancer research?

ADORA2A plays critical roles in immunomodulation within the tumor microenvironment, making ADORA2A antibodies valuable tools for cancer immunology research:

Applications in Cancer Immunology Research:

Future research directions should focus on integrating ADORA2A-targeted approaches with existing immunotherapies, as ADORA2A regulation may influence response to checkpoint inhibitors and other immunomodulatory treatments.

What emerging techniques show promise for studying ADORA2A-mediated signaling in neurological disorders?

Emerging techniques for studying ADORA2A-mediated signaling in neurological disorders combine advanced molecular tools with sophisticated imaging approaches:

Cutting-Edge Techniques for ADORA2A Neurological Research:

• Cell-Type Specific Manipulation:

  • CRISPR-Cas9 mediated conditional knockout systems for precise temporal and spatial control of ADORA2A

  • Viral vector-mediated expression of ADORA2A in specific neuronal populations

  • These approaches allow for dissection of ADORA2A's role in different neural circuits

• Advanced Imaging Methodologies:

  • Super-resolution microscopy to visualize ADORA2A distribution at synapses

  • Fiber photometry with genetically encoded calcium indicators in ADORA2A-expressing neurons

  • These techniques provide insights into how ADORA2A modulates dopamine and glutamate signaling, which is crucial for neural balance

• Single-Cell Transcriptomics:

  • Correlate ADORA2A expression with transcriptional profiles at single-cell resolution

  • Identify cell-specific signaling pathways and interacting partners

  • These approaches have revealed heterogeneity in ADORA2A-expressing neuronal populations

• Functional Circuit Mapping:

  • Chemogenetic and optogenetic tools targeted to ADORA2A-expressing neurons

  • Simultaneous electrophysiological recording and ADORA2A imaging

  • These methodologies help understand how ADORA2A contributes to network-level functions

• Translational Biomarker Development:

  • PET imaging with ADORA2A-specific ligands

  • Correlation of cerebrospinal fluid adenosine levels with clinical outcomes

  • These approaches bridge preclinical models and clinical applications

ADORA2A's critical role in modulating dopamine and glutamate signaling makes it particularly relevant for studying disorders like Parkinson's disease, where adenosine-dopamine interactions in the striatum influence motor control . The combined use of these emerging techniques promises to provide unprecedented insights into ADORA2A's complex roles in neurological function and dysfunction.

What methodological considerations are important when studying ADORA2A's epigenetic regulation across different disease contexts?

Epigenetic regulation of ADORA2A varies across disease contexts, requiring specialized methodological approaches for comprehensive characterization:

Key Methodological Considerations for ADORA2A Epigenetic Studies: