CCR7 Antibody, FITC conjugated

What is CCR7 and why is it significant in immunological research?

CCR7 (CD197) is a G-protein-coupled chemokine receptor with seven membrane-spanning domains that functions as a receptor for chemokines including 6Ckine/SLC, CCL19, and CCL21 . Its significance stems from its critical role in lymphocyte trafficking, particularly T cell migration to secondary lymphoid organs. CCR7 expression helps distinguish between naive and memory T cell populations, making it an essential marker for immunophenotyping . The receptor was originally identified as being induced by Epstein-Barr virus (EBV) and is thought to mediate EBV effects on B lymphocytes . Its expression pattern in blood, bone marrow, lymph nodes, and intestinal tissues makes it particularly valuable for studying immune cell migration and function in various anatomical compartments .

What cell types express CCR7 and how can they be identified using CCR7 antibodies?

CCR7 is predominantly expressed on:

• T lymphocytes (particularly naive and central memory T cells)

• B lymphocytes (especially after activation)

• Dendritic cells (with increasing expression during maturation)

These populations can be identified using flow cytometry with CCR7 antibodies in combination with other lineage markers. For T cells, CCR7 and CD62L expression patterns help differentiate between naive (CCR7+CD62L+), central memory (CCR7+CD62L+), and effector memory (CCR7-CD62L-) subsets . In a typical experiment, human peripheral blood lymphocytes can be stained with anti-CD4 and anti-CCR7 antibodies to visualize distinct T cell subpopulations . Studies have demonstrated that a major population of CD4 or CD8 T cells expressing CCR7 is also CD62L positive, confirming the utility of CCR7 antibodies in identifying specific T cell subsets .

What are the recommended staining protocols for CCR7 antibody (FITC conjugated)?

For effective CCR7 staining:

• Prepare single cell suspensions from tissues or blood samples

• Block Fc receptors to prevent non-specific binding

• Add 5 μL (0.5 μg) of FITC-conjugated CCR7 antibody per test (defined as the amount needed to stain a cell sample in a final volume of 100 μL)

• Incubate for at least 45 minutes at 2-8°C (extended incubation is crucial for optimal staining)

• Wash cells to remove unbound antibody

• Analyze by flow cytometry using appropriate instrument settings for FITC detection

Cell numbers can range from 10^5 to 10^8 cells per test, though the optimal concentration should be determined empirically for each application . For multicolor applications, include appropriate single-stained controls for compensation and fluorescence-minus-one (FMO) controls to establish proper gating strategies.

How can researchers distinguish between different memory T cell subsets using CCR7 antibodies?

Distinguishing T cell memory subsets requires a sophisticated understanding of CCR7 expression patterns in combination with other markers. The CCR7 antibody is instrumental in separating central memory T cells (TCM) from effector memory T cells (TEM) . Research has shown that in murine models, CD4 or CD8 T cells can be subdivided based on CCR7, CD62L, and CD44 expression .

Flow cytometric analysis has demonstrated that activated naive CD4 T cells only downregulate CCR7 after multiple cell divisions, coinciding with CD62L downregulation and increased production of cytokines like IL-4 and IFN-γ . During secondary immune responses, the majority of IL-2 and IFN-γ-producing cells are CCR7 low, while few cytokine-expressing CCR7 high T cells are detected . This marker combination allows researchers to track the differentiation of T cells during immune responses and correlate phenotype with functionality.

What methodological considerations should be addressed when investigating CCR7 expression during dendritic cell maturation?

Dendritic cell (DC) maturation is accompanied by significant changes in CCR7 expression that enable migration to lymphoid tissues. When studying this process:

• Maturation stimuli selection: Different maturation stimuli (LPS, cytokines, immune complexes) may induce varying levels of CCR7 expression. For instance, immune complex stimulation through FcγR engagement can stimulate DC migration from peripheral tissues to lymph node paracortex in a CCR7-dependent manner .

• Temporal dynamics: CCR7 expression increases progressively during DC maturation, necessitating time-course experiments to capture expression kinetics.

• Functional correlation: Combine CCR7 expression analysis with functional migration assays using three-dimensional matrices and CCR7 ligands (CCL19/CCL21) to correlate expression with migratory capacity .

• Signaling pathway analysis: Consider the involvement of specific signaling pathways (PI3K, ERK) that regulate CCR7 expression and function. Studies have shown that immune complex-induced responses can be modulated by ERK inhibition .

• Confounding factors: FcγR engagement and subsequent signaling can influence CCR7 expression and function. When using antibody-based detection methods, proper FcγR blocking is essential to prevent experimental artifacts .

These considerations are crucial for researchers investigating the role of CCR7 in dendritic cell biology, particularly in the context of vaccine development, autoimmunity, and tumor immunology.

How can researchers accurately interpret CCR7 internalization dynamics following ligand binding?

CCR7 undergoes internalization upon ligand binding, which can complicate experimental interpretation. Studies have shown that CCR7 is internalized via clathrin-coated pits, with the majority recycled back to the plasma membrane . When investigating receptor internalization:

• Distinguish surface from intracellular expression: Use differential staining protocols (surface staining versus permeabilized cells) to quantify the proportion of internalized versus surface-expressed CCR7.

• Temporal resolution: Design time-course experiments that capture rapid internalization events, typically occurring within minutes of ligand exposure.

• Ligand specificity: Different CCR7 ligands (CCL19 versus CCL21) may induce varying internalization kinetics and downstream signaling events.

• Recycling dynamics: Account for receptor recycling when interpreting results, as many internalized CCR7 molecules return to the cell surface rather than undergoing degradation.

• Signal transduction correlation: Correlate internalization with downstream signaling events to distinguish between signal termination and signal propagation from endosomal compartments.

These methodological considerations are critical for accurately interpreting experiments investigating CCR7 trafficking and signaling, particularly in studies of T cell and dendritic cell migration.

What are the experimental challenges in detecting CCR7 expression on rare cell subsets?

Detecting CCR7 on rare cell populations presents several technical challenges:

• Signal-to-noise optimization: FITC-conjugated antibodies may not provide sufficient signal-to-noise ratio for rare populations. Consider:

  • Extended incubation periods (minimum 45 minutes at 2-8°C) for optimal staining

  • Alternative brighter fluorophores for rare population analysis

  • Signal amplification techniques when necessary

• Pre-enrichment strategies: For extremely rare populations, consider magnetic pre-enrichment before flow cytometry analysis.

• Multiparameter approach: Combine CCR7 with additional markers to accurately identify rare subsets within heterogeneous samples.

• Proper controls: Include biological controls (CCR7-deficient cells or CCR7-transfected cell lines) to establish specificity . For example, comparisons between HEK293 cells transfected with human CCR7 versus irrelevant transfectants can validate antibody specificity .

• Live/dead discrimination: Include viability dyes to exclude dead cells that may bind antibodies non-specifically.

• Standardization: Use quantitative beads to standardize fluorescence intensity across experiments and instruments.

These approaches can significantly improve the reliability of CCR7 detection on rare cellular subsets, particularly in complex samples like peripheral blood or tissue digests.

How should researchers optimize flow cytometry panels that include CCR7-FITC antibodies?

Optimizing multicolor flow cytometry panels containing CCR7-FITC requires careful consideration of several factors:

• Fluorophore selection: FITC emits at 520 nm following 488 nm excitation . When designing panels:

  • Avoid combining with PE-Texas Red or PerCP, which have spectral overlap

  • Consider brightness hierarchy, placing FITC on highly expressed antigens

  • Use brighter fluorophores (PE, APC) for low-density antigens

• Antibody titration: Determine the optimal concentration experimentally. Starting with 5 μL (0.5 μg) per test is recommended, but titration is essential to maximize signal-to-noise ratio .

• Compensation setup: Include single-stained controls for each fluorophore to properly compensate for spectral overlap.

• Protocol adaptation: Extend CCR7 staining incubation to at least 45 minutes at 2-8°C for optimal results .

• Biological controls: Include CCR7-negative and CCR7-positive populations to validate staining specificity. Lymphocyte subsets with known CCR7 expression patterns serve as internal controls.

• Data analysis strategy: Implement a consistent gating strategy that accounts for cell size, granularity, and viability before analyzing CCR7 expression.

Adhering to these optimization principles ensures reliable and reproducible CCR7 detection in multiparameter flow cytometry experiments.

What are the key technical differences between monoclonal and polyclonal CCR7 antibodies?

The choice between monoclonal and polyclonal CCR7 antibodies significantly impacts experimental outcomes:

For example, the 3D12 monoclonal antibody has been specifically validated for flow cytometric analysis of human peripheral blood cells , while polyclonal antibodies might offer advantages in detecting CCR7 across multiple species or in tissues where epitope accessibility may be limited . Researchers should select the appropriate antibody type based on their specific experimental requirements and the level of characterization needed.

How can researchers validate CCR7 antibody specificity in their experimental systems?

Validating CCR7 antibody specificity is crucial for reliable data interpretation:

• Positive and negative controls:

  • Use CCR7-transfected cell lines as positive controls

  • Include CCR7-deficient cells (from knockout mice) as negative controls

  • Compare staining between cell populations with known differential CCR7 expression

• Blocking experiments:

  • Pre-incubate cells with unlabeled CCR7 antibody before adding FITC-conjugated antibody

  • Observe competitive inhibition of staining

• Functional validation:

  • Correlate CCR7 staining with migratory responses to CCR7 ligands (CCL19/CCL21)

  • Verify that migration is inhibited by CCR7 neutralization

• Specificity testing in transfected systems:

  • Compare staining between HEK293 cells transfected with human CCR7 and irrelevant transfectants

  • Confirm absence of non-specific binding to irrelevant receptors

• Cross-platform validation:

  • Compare flow cytometry results with other detection methods (e.g., RT-PCR, western blot)

  • Ensure concordance between protein and mRNA expression

These validation approaches ensure that experimental observations genuinely reflect CCR7 biology rather than technical artifacts.

What considerations are important when comparing human and murine CCR7 expression patterns?

Comparative studies of CCR7 across species require careful methodological considerations:

• Antibody selection: Choose antibodies with validated cross-reactivity or species-specific antibodies. While some antibodies recognize conserved epitopes, many are species-specific .

• Expression pattern differences:

  • Human CCR7 (UniProt ID: P32248)

  • Mouse CCR7 (UniProt ID: P47774)

  • Sequence homology is high but not identical, potentially affecting antibody binding

• Functional conservation: Though structurally similar, species differences in ligand binding affinities, internalization kinetics, and downstream signaling may exist.

• Technical approach harmonization: When comparing across species, standardize:

  • Sample preparation methods

  • Antibody concentrations (normalized to epitope density)

  • Instrument settings and analysis parameters

• Validation in each species: Validate antibody specificity independently in each species using appropriate controls:

  • Species-specific CCR7-deficient cells

  • CCR7-transfected cell lines from the relevant species

These considerations are particularly important for translational research attempting to extrapolate findings from murine models to human immunology.

How can CCR7 expression analysis contribute to understanding T cell differentiation kinetics?

CCR7 expression dynamics provide valuable insights into T cell differentiation:

• Temporal profiling: Studies have shown that activated naive CD4 T cells downregulate CCR7 only after multiple cell divisions, coinciding with CD62L downregulation and cytokine production . This allows researchers to track progressive differentiation stages.

• Correlation with functional acquisition: During secondary immune responses, IL-2 and IFN-γ production is predominantly associated with CCR7-low cells, while few cytokine-expressing CCR7-high T cells are detected . This correlation helps define functional maturation stages.

• Integration with division tracking: Combining CCR7 staining with proliferation dyes (CFSE, CellTrace) enables correlation between division history and phenotypic changes.

• Mathematical modeling: CCR7 expression kinetics can be incorporated into mathematical models of T cell differentiation to predict population dynamics during immune responses.

• Single-cell approaches: Pairing CCR7 detection with single-cell RNA sequencing or mass cytometry creates high-dimensional datasets that reveal differentiation trajectories and transitional states.

This multifaceted analysis of CCR7 expression provides a framework for understanding the progressive development of effector and memory T cell populations during immune responses.

What are the best practices for analyzing CCR7-dependent migration in complex tissue environments?

Analyzing CCR7-dependent migration requires sophisticated approaches:

• Three-dimensional migration assays: Use 3D matrices rather than traditional transwell systems to better recapitulate tissue architecture . This approach more accurately reflects the complex environment cells navigate in vivo.

• Live imaging techniques: Employ time-lapse microscopy with fluorescently labeled cells to track:

  • Directionality parameters (straightness index, turning angles)

  • Velocity components (speed, persistence)

  • Interaction dynamics with tissue structures

• Chemokine gradient characterization: Quantify CCL19/CCL21 gradients using immunofluorescence or protein concentration measurements to correlate migration with actual chemokine landscapes.

• Integrated analysis of CCR7 expression and function:

  • Correlate surface CCR7 levels with migratory capacity

  • Assess the impact of receptor internalization on sustained directional movement

  • Evaluate the role of receptor recycling in maintaining migratory responses

• Control experiments:

  • Use CCR7-deficient cells as negative controls

  • Include perturbation conditions (receptor blockade, signaling inhibitors)

  • Compare wild-type and FcγRIIb-deficient dendritic cells, which show differential migration patterns

These approaches provide comprehensive insights into how CCR7 governs cellular navigation through complex tissue environments.

How are emerging technologies enhancing CCR7 expression analysis in immunological research?

Recent technological advances are transforming CCR7 research:

• High-parameter cytometry: Spectral flow cytometry and mass cytometry (CyTOF) enable simultaneous detection of CCR7 alongside dozens of other markers, providing unprecedented phenotypic resolution.

• Single-cell omics: Integration of CCR7 protein expression data with single-cell transcriptomics and epigenomics reveals regulatory networks governing expression and function.

• Advanced imaging:

  • Super-resolution microscopy visualizes CCR7 distribution and clustering on the cell membrane

  • Intravital imaging tracks CCR7-dependent migration in living organisms

• Engineered reporters:

  • CRISPR knock-in fluorescent proteins to endogenous CCR7 loci

  • Biosensors that detect CCR7 activation and downstream signaling

• AI-assisted analysis: Machine learning algorithms identify subtle CCR7 expression patterns and correlations with functional outcomes from complex datasets.

These emerging technologies promise to deepen our understanding of CCR7 biology in health and disease, potentially leading to new therapeutic strategies targeting this important chemokine receptor.