CCR2 Antibody

What is CCR2 and why is it important in immunological research?

CCR2 is a G-protein coupled receptor with seven transmembrane domains that preferentially binds monocyte chemoattractant proteins (MCP-1/CCL2 and MCP-3/CCL7). In humans, the canonical protein consists of 374 amino acid residues with a mass of 41.9 kDa and is primarily localized in the cell membrane . CCR2 plays essential roles in chemotaxis and cytokine-mediated signaling pathways, functioning as a key receptor in monocyte migration, T-cell differentiation, and inflammatory responses . The receptor exists in two isoforms (CCR2A and CCR2B) that arise from alternative splicing and differ only at their intracellular carboxyl terminals, with CCR2A possessing 14 additional amino acids . This structural difference may provide a mechanism for activating different intracellular second messengers in response to similar extracellular ligands, making CCR2 an important target in inflammation, infection, and disease research .

Which cell types express CCR2 and how can antibodies help identify them?

CCR2 is predominantly expressed by:

• Monocytes and macrophages

• T cells

• Natural killer (NK) cells

• Basophils

• Mast cells

• Dendritic cells

• Some reports suggest B cells may also express CCR2 receptors

Anti-CCR2 antibodies are valuable tools for identifying these cell populations in flow cytometry and immunohistochemistry. For instance, in flow cytometry applications, researchers can use anti-CCR2 antibodies in combination with other surface markers like CD14 (for monocytes) to identify specific CCR2-expressing populations . This enables phenotypic characterization of cell subsets involved in inflammatory responses and can help track cellular migration patterns in disease models .

How do I choose between monoclonal and polyclonal CCR2 antibodies?

The selection between monoclonal and polyclonal anti-CCR2 antibodies depends on your specific research application:

Monoclonal antibodies (e.g., clone EPR20844-15, clone 48607):

• Provide consistent batch-to-batch reproducibility

• Offer high specificity for a single epitope

• Ideal for quantitative applications requiring precision

• Better for distinguishing between closely related proteins

• Recommended for flow cytometry, where specific epitope recognition is critical

Polyclonal antibodies:

• Recognize multiple epitopes on the CCR2 protein

• May provide stronger signals by binding multiple sites

• More tolerant of protein denaturation in applications like Western blotting

• Potentially more robust across diverse sample types

• Useful when protein conformation might be altered

For critical applications requiring epitope specificity, validated monoclonal antibodies like those with knockout validation would be preferable . For applications where sensitivity is paramount, polyclonal antibodies may offer advantages through their ability to bind multiple epitopes.

What are the optimal methods for detecting CCR2 in human samples using antibodies?

The detection of CCR2 in human samples can be accomplished through several techniques, each with specific methodological considerations:

Flow Cytometry:

• Particularly effective for cell surface CCR2 detection on intact cells

• Requires careful consideration of staining buffers to preserve receptor conformation

• Typically uses live, non-fixed cells to maintain membrane integrity

• Example protocol: Stain peripheral blood monocytes with anti-CCR2 antibody (e.g., MAB150) followed by fluorophore-conjugated secondary antibody

• Include proper isotype controls to account for non-specific binding

• Consider dual staining with lineage markers (e.g., CD14 for monocytes) to identify specific CCR2+ populations

Immunohistochemistry (IHC):

• Allows visualization of CCR2 distribution in tissue context

• May require antigen retrieval optimization to expose membrane-bound epitopes

• Antibodies validated for IHC applications should be selected

• Recombinant antibody formats like EPR20844-15 provide consistent lot-to-lot performance

Western Blotting:

• Useful for quantifying total CCR2 protein expression

• Sample preparation is critical - membrane proteins require specific lysis buffers

• Expected band size is approximately 41.9 kDa, though post-translational modifications may alter migration patterns

• Consider both reducing and non-reducing conditions as protein conformation may affect antibody recognition

How can I optimize antibody-based detection of CCR2 in flow cytometry experiments?

Flow cytometry is commonly used for CCR2 detection on immune cells, but requires specific optimization:

• Sample preparation:

  • Use freshly isolated cells whenever possible

  • For peripheral blood mononuclear cells (PBMCs), process samples within 4-6 hours of collection

  • Avoid harsh fixation protocols that may disrupt membrane protein epitopes

• Staining protocol optimization:

  • Perform staining at 4°C to prevent receptor internalization

  • Include sodium azide in staining buffers to inhibit metabolic processes

  • Consider using Fc receptor blocking reagents to reduce non-specific binding

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

• Controls and validation:

  • Always include appropriate isotype controls (e.g., Mouse IgG2B for clone 48607)

  • Include fluorescence-minus-one (FMO) controls to set accurate gates

  • Validate staining patterns with known positive populations (e.g., monocytes) and negative populations

• Analysis considerations:

  • Gate on live cells first to exclude non-specific binding to dead cells

  • Consider CCR2 expression levels in relation to other markers (e.g., CD14+CCR2+ for classical monocytes)

  • Be aware that CCR2 expression may be altered during inflammatory states or disease conditions

What are the common pitfalls when using CCR2 antibodies and how can they be avoided?

Several technical challenges can arise when using CCR2 antibodies:

• Receptor internalization:

  • CCR2 can rapidly internalize upon ligand binding or cell activation

  • Perform staining at 4°C and include sodium azide in buffers

  • Consider fixation immediately after staining to "freeze" receptor location

• Epitope masking:

  • Glycosylation and other post-translational modifications may mask epitopes

  • Test multiple antibody clones recognizing different epitopes

  • For certain applications, consider enzymatic treatment to remove glycosylation

• Cross-reactivity:

  • CCR2 shares structural similarities with other chemokine receptors

  • Use antibodies validated for specificity, ideally with knockout validation

  • Include appropriate controls in multiparameter experiments

• Signal variability:

  • Expression levels can vary with cell activation state

  • Standardize sample collection and processing times

  • Consider using stabilizing fixatives for consistent results

• Species specificity:

  • Ensure the antibody is validated for your species of interest

  • Cross-reactivity between human and rodent CCR2 varies between antibody clones

How can CCR2 antibodies be used to investigate CCR2's role in disease pathogenesis?

CCR2 antibodies enable sophisticated investigations into disease mechanisms:

In inflammatory diseases:

• Quantify CCR2+ cell infiltration in tissue sections using immunohistochemistry

• Measure changes in CCR2 expression levels on specific immune cell populations

• Track CCR2+ cell migration in response to inflammatory stimuli

• Correlate CCR2 expression with disease severity and progression

In infectious diseases (e.g., COVID-19):

• Recent research demonstrates that CCR2 is upregulated in COVID-19 patients at the mRNA level in peripheral blood mononuclear cells

• CCR2 antibodies can be used to characterize the immune cell subsets responding to infection

• Flow cytometric analysis can reveal how infection alters CCR2 expression patterns

• Studies have shown significant differences in CCR2 expression between COVID-19 patients and healthy controls, particularly in individuals over 60 years of age (P = 0.0353)

In cancer research:

• Examine tumor-associated macrophage populations for CCR2 expression

• Investigate how CCR2+ cells contribute to tumor microenvironment

• Analyze CCR2 expression in relation to cancer progression and metastasis

What are the considerations for using CCR2 antibodies in multiplex immunofluorescence assays?

Multiplex immunofluorescence allows simultaneous detection of multiple markers including CCR2:

• Antibody panel design:

  • Select anti-CCR2 antibodies with minimal spectral overlap with other fluorophores

  • Consider the relative expression levels of targets when assigning fluorophores

  • Test for potential antibody cross-reactivity or steric hindrance

• Sequential staining approaches:

  • For membrane proteins like CCR2, consider staining order carefully

  • Test whether CCR2 staining is affected by fixation needed for intracellular targets

  • Validate multiplex panels on known positive and negative controls

• Signal amplification strategies:

  • For weak CCR2 expression, consider tyramide signal amplification

  • Evaluate whether amplification affects other markers in the panel

  • Titrate primary antibody concentrations carefully when using amplification

• Image analysis considerations:

  • Account for membrane localization when setting analysis parameters

  • Establish clear thresholds for positive CCR2 staining

  • Consider colocalization analyses with other membrane markers

How can researchers evaluate the functional effects of CCR2 blockade using antibodies?

Blocking antibodies against CCR2 can be powerful tools for functional studies:

• In vitro migration assays:

  • Transwell migration assays using CCL2 as chemoattractant

  • Pre-incubation with anti-CCR2 blocking antibodies

  • Quantification of migration inhibition as functional readout

  • Dose-response titration to determine IC50 values

• Receptor signaling studies:

  • Measure phosphorylation of downstream mediators (e.g., PI3K, Rac)

  • Investigate CCR2-mediated calcium flux with and without antibody blockade

  • Examine effects on lamellipodium protrusion in real-time imaging

• In vivo models:

  • Administration of blocking antibodies in disease models (e.g., inflammatory conditions)

  • Analysis of monocyte/macrophage infiltration in tissues

  • Assessment of disease parameters in the presence of CCR2 blockade

  • Evaluation of potential therapeutic effects in preclinical models

How are CCR2 antibodies being used to investigate the CCL2/CCR2 axis as a therapeutic target?

The CCL2/CCR2 signaling pathway is emerging as a promising therapeutic target in various diseases:

Inflammatory diseases:

• Anti-CCR2 antibodies can be used to characterize receptor expression in patient samples

• Flow cytometric analysis of CCR2+ inflammatory monocytes can serve as a biomarker

• Immunohistochemical evaluation of tissues can reveal CCR2+ cell infiltration patterns

• Correlation of CCR2 expression with treatment response may identify patient subsets

COVID-19 research:

• Studies have demonstrated that both CCL2 (at protein level) and CCR2 (at mRNA level) are upregulated in COVID-19 patients

• CCL2 serum levels were significantly elevated in patients compared to healthy controls across all age groups (P < 0.0001)

• CCR2 expression was higher in COVID-19 patients, with significant differences observed in individuals over 60 years of age (P = 0.0353)

• These findings suggest that targeting the CCL2/CCR2 axis could be a potential therapeutic approach for improving COVID-19 patient outcomes

Neurodegenerative diseases:

• CCR2 plays important roles in mediating peripheral nerve injury-induced neuropathic pain

• Research indicates CCR2 increases NMDA-mediated synaptic transmission in both dopamine D1 and D2 receptor-containing neurons, potentially through MAPK/ERK-dependent phosphorylation mechanisms

• Antibodies can help characterize these mechanisms in tissue samples

What are the considerations for using CCR2 antibodies in single-cell analysis technologies?

Single-cell technologies present unique opportunities and challenges for CCR2 detection:

Single-cell RNA sequencing (scRNA-seq):

• While not using antibodies directly, scRNA-seq data can guide subsequent protein validation

• CCR2 transcript levels can identify cell populations for targeted antibody validation

• Integration of transcriptomic and proteomic data requires careful analysis of correlation patterns

Mass cytometry (CyTOF):

• Requires metal-conjugated anti-CCR2 antibodies

• Allows simultaneous detection of many markers without fluorescence spectral overlap

• Antibody clone selection is critical as fixation requirements may affect epitope recognition

• Validation against flow cytometry standards is recommended

Imaging mass cytometry:

• Enables spatial analysis of CCR2+ cells in tissue context

• Requires antibodies validated for formalin-fixed paraffin-embedded tissues

• Multiplexing capabilities allow correlation with tissue microenvironment features

How can researchers address CCR2 isoform specificity with antibodies?

CCR2 exists in two splice variants (CCR2A and CCR2B) that differ in their C-terminal domains:

Isoform selectivity:

• Most commercial antibodies recognize both CCR2A and CCR2B

• For isoform-specific detection, consider antibodies targeting the C-terminal region

• Custom antibodies against the unique 14 amino acids of CCR2A may be required

• Validate isoform specificity using recombinant expression systems

Experimental approaches:

• Combine antibody detection with PCR-based isoform quantification

• Use isoform-specific siRNA knockdown to validate antibody specificity

• Consider using epitope-tagged isoform constructs for overexpression studies

Functional implications:

• Different CCR2 isoforms may activate distinct signaling pathways

• Isoform-specific antibodies can help determine their relative contributions

• Blocking studies may reveal isoform-specific functions in cellular responses

What validation methods should researchers use to confirm CCR2 antibody specificity?

Rigorous validation is essential for reliable CCR2 antibody applications:

Genetic validation approaches:

• Testing on CCR2 knockout cell lines or tissues is the gold standard

• siRNA or shRNA knockdown of CCR2 followed by antibody staining

• Overexpression systems comparing transfected vs. non-transfected cells

Comparative validation:

• Testing multiple antibody clones against the same samples

• Comparing antibody detection with mRNA expression data

• Cross-validation using different detection techniques (e.g., flow cytometry vs. Western blot)

Epitope competition:

• Blocking with recombinant peptides containing the target epitope

• Dose-dependent reduction in signal should be observed

• Negative control peptides should not affect antibody binding

Application-specific controls:

• For flow cytometry: Isotype controls, fluorescence-minus-one controls

• For IHC: Absorption controls, secondary-only controls

• For Western blotting: Molecular weight verification, positive and negative control lysates

How do different fixation and permeabilization methods affect CCR2 antibody binding?

Membrane proteins like CCR2 are particularly sensitive to fixation conditions:

Fixation considerations:

• Paraformaldehyde (1-4%): Generally preserves CCR2 epitopes but may cause some conformational changes

• Methanol/acetone: May denature membrane proteins, potentially destroying conformational epitopes

• Glutaraldehyde: Generally too harsh for most CCR2 epitopes

• Optimal fixation duration is typically brief (10-15 minutes) to preserve epitope integrity

Permeabilization effects:

• Saponin (0.1-0.5%): Gentle detergent that often preserves membrane protein structure

• Triton X-100: More harsh, may disrupt membrane protein conformation

• Digitonin: Selective permeabilization of plasma membrane, useful for distinguishing surface from intracellular CCR2

Protocol recommendations:

• For flow cytometry: Consider staining for CCR2 before fixation when possible

• For microscopy: Test multiple fixation protocols on known positive samples

• For tissue sections: Optimize antigen retrieval methods specifically for CCR2

What are the best practices for quantifying CCR2 expression levels using antibodies?

Accurate quantification of CCR2 requires standardized approaches:

Flow cytometry quantification:

• Use calibration beads with known antibody binding capacity

• Convert fluorescence intensity to antibodies bound per cell (ABC)

• Establish consistent gating strategies based on appropriate controls

• Report both percentage of positive cells and median fluorescence intensity

Western blot quantification:

• Include recombinant CCR2 protein standards for calibration curve

• Use housekeeping proteins appropriate for membrane fraction normalization

• Consider phosphorylation-state specific quantification for activation studies

• Employ digital image analysis with linear dynamic range

Immunohistochemistry quantification:

• Develop consistent scoring systems (e.g., H-score, Allred score)

• Use digital pathology software for unbiased quantification

• Include internal control tissues in each staining batch

• Consider multiplex approaches to normalize to cell numbers