ALCAM (CD166) Mouse

What is ALCAM/CD166 and what is its molecular structure in mice?

ALCAM, also known as CD166, is a type I membrane glycoprotein belonging to the immunoglobulin supergene family. In mice, ALCAM spans from amino acids Trp28-Lys527 (based on accession # AAC06342) and shares significant homology with human ALCAM. The protein contains multiple immunoglobulin domains and functions as a cell adhesion molecule with both homotypic (self-binding) and heterotypic binding capabilities. As a membrane-bound glycoprotein, mouse ALCAM appears as a 90-120 kDa protein in Western blot analysis under reducing conditions .

Where is ALCAM/CD166 expressed in mouse tissues?

In mice, ALCAM is primarily expressed on thymic epithelial cells, activated B and T cells, and monocytes. Expression can be detected in mouse brain tissue, particularly in the cerebellum, as demonstrated by Western blot analysis . Flow cytometry analysis shows differential expression on activated versus resting mouse splenocytes, with increased expression observed following activation . This expression pattern suggests important roles in both the immune and nervous systems, with potential implications for T cell development, immune regulation, and neurological functions .

How conserved is mouse ALCAM compared to its human homolog?

Comparison of mouse ALCAM with human ALCAM reveals significant conservation, particularly in the CD6-binding domain, which mediates cross-species binding interactions. This conservation makes mouse models valuable for studying ALCAM-related mechanisms with potential translational relevance to human biology. The conservation of the CD6-binding domain across different species demonstrates the evolutionary importance of ALCAM-CD6 interactions in immune function .

What antibodies are available for mouse ALCAM/CD166 detection?

Several validated antibodies are available for detecting mouse ALCAM/CD166:

• PE-conjugated antibodies (e.g., FAB1172P) - Optimized for flow cytometry applications, allowing direct detection of ALCAM on cell surfaces without secondary antibody requirements .

• Non-conjugated antibodies (e.g., AF1172) - Suitable for multiple applications including Western blot and flow cytometry (with appropriate secondary antibodies). These antibodies show cross-reactivity with human, mouse, rat, and canine ALCAM/CD166, making them versatile for comparative studies .

Both antibody types have been validated in flow cytometry applications with mouse splenocytes and in Western blot analyses with mouse brain tissue .

What are the optimal flow cytometry protocols for analyzing ALCAM expression in mouse immune cells?

For optimal flow cytometry analysis of ALCAM expression in mouse immune cells:

• Sample preparation: Use freshly isolated mouse splenocytes (either resting or activated depending on research question).

• Staining approach: For direct detection, use PE-conjugated anti-ALCAM antibodies (e.g., FAB1172P at manufacturer-recommended concentrations). For indirect detection, use primary anti-ALCAM antibodies (e.g., AF1172) followed by fluorophore-conjugated secondary antibodies.

• Multi-parameter analysis: Combine ALCAM staining with other markers such as B220/CD45R (for B cells) to identify ALCAM expression on specific cell populations.

• Controls: Set quadrant markers based on appropriate isotype control antibodies (e.g., IC108P) to ensure accurate gating and analysis.

• Analysis: Compare expression patterns between activated and resting cells to assess activation-dependent changes in ALCAM expression .

What methods can be used to detect soluble versus membrane-bound ALCAM in mouse samples?

Researchers can differentiate between soluble and membrane-bound ALCAM using complementary approaches:

• For soluble ALCAM:

  • ELISA assays to quantify ALCAM levels in mouse serum or culture supernatants

  • Western blot of precipitated serum proteins

  • Functional binding assays with recombinant CD6

• For membrane-bound ALCAM:

  • Flow cytometry of intact cells using anti-ALCAM antibodies

  • Immunohistochemistry or immunofluorescence of tissue sections

  • Cell surface biotinylation followed by precipitation and Western blot

• For comparative analysis:

  • Measure ALCAM mRNA expression via RT-PCR to compare with protein levels

  • Analyze ALCAM shedding in response to various stimuli by monitoring changes in soluble versus membrane-bound ratios .

How does ALCAM regulate T cell responses in mouse models?

ALCAM plays a critical role in regulating T cell responses through its interaction with CD6, a co-stimulatory molecule expressed on T cells. This interaction facilitates long-term contact between antigen-presenting cells and T cells, influencing immune response development. In studies using ALCAM-deficient mice:

• T cell proliferation is significantly reduced, including total cells, CD3+CD4+ T cells, and activated T cells in immune tissues.

• The interaction between ALCAM on dendritic cells and CD6 on T cells promotes T cell activation, as demonstrated by reduced proliferation when co-cultured T cells and dendritic cells are treated with anti-CD6 antibodies.

• Adoptive transfer experiments show that wild-type mice sensitized with OVA-pulsed ALCAM-deficient bone marrow-derived dendritic cells exhibit reduced immune responses compared to those receiving wild-type dendritic cells.

These findings collectively demonstrate that ALCAM-CD6 interactions are critical for optimal T cell activation and proliferation in mouse models .

What is the role of ALCAM in mouse models of food allergy?

In ovalbumin (OVA)-induced food allergy mouse models, ALCAM has been shown to significantly influence allergic responses:

• Serum ALCAM levels increase while mRNA expression decreases in OVA-challenged wild-type mice, suggesting increased shedding of membrane-bound ALCAM during allergic responses.

• ALCAM-deficient (ALCAM−/−) mice show attenuated responses to OVA challenge compared to wild-type mice, including:

  • Reduced serum IgE levels

  • Decreased Th2 cytokine mRNA expression

  • Diminished histological injuries associated with food allergy

  • Reduced T cell proliferation in immune tissues

• The attenuation of immune responses in ALCAM-deficient mice suggests that ALCAM plays a crucial role in mediating allergic immune responses, particularly by facilitating T helper type 2 (Th2) cell activation and proliferation.

These findings indicate that ALCAM is a potential therapeutic target for food allergy and other Th2-mediated allergic conditions .

How do ALCAM-CD6 interactions influence T cell development in mice?

ALCAM-CD6 interactions play important roles in T cell development:

• ALCAM expressed on thymic epithelial cells interacts with CD6 on developing thymocytes, potentially influencing selection processes during T cell maturation.

• This interaction may contribute to the establishment of the T cell repertoire by affecting positive and negative selection thresholds.

• The conserved nature of the CD6-binding domain across species suggests evolutionary importance in T cell development processes.

• In ALCAM-deficient mice, alterations in T cell development and maturation may contribute to the observed changes in immune responses to challenges such as allergens.

The specific mechanisms through which ALCAM-CD6 interactions regulate T cell development require further investigation using thymic organ cultures and detailed analysis of T cell subpopulations in ALCAM-deficient versus wild-type mice .

How can researchers effectively generate and validate ALCAM-deficient mouse models?

Generating and validating ALCAM-deficient mouse models requires several methodological steps:

• Generation approaches:

  • CRISPR/Cas9 gene editing targeting the ALCAM gene

  • Traditional gene knockout approaches using homologous recombination

  • Conditional knockout systems using Cre-loxP for tissue-specific deletion

• Validation methods:

  • Genotyping to confirm genetic modification

  • RT-PCR to verify absence of ALCAM mRNA

  • Western blot analysis of tissue lysates (particularly brain and immune tissues) to confirm absence of protein

  • Flow cytometry of immune cells (especially splenocytes) to verify lack of surface expression

  • Functional assays to assess immune responses in various challenges

• Phenotypic characterization:

  • Analysis of lymphoid organ development

  • T cell subpopulation assessment

  • Response to immunological challenges (e.g., OVA sensitization)

  • Histological examination of relevant tissues

This comprehensive approach ensures the generation of reliable ALCAM-deficient models for investigating ALCAM's functions in various physiological and pathological contexts .

What experimental approaches are most effective for studying ALCAM-CD6 interactions in mouse models?

To effectively study ALCAM-CD6 interactions in mouse models, researchers can employ:

• Binding assays:

  • Surface plasmon resonance using recombinant mouse ALCAM and CD6 proteins

  • Cell adhesion assays with ALCAM or CD6 expressing cells

  • Fluorescence resonance energy transfer (FRET) to detect molecular proximity

• Functional studies:

  • Co-cultures of dendritic cells and T cells from wild-type and ALCAM-deficient mice

  • Blocking studies using anti-CD6 or anti-ALCAM antibodies

  • Adoptive transfer experiments with ALCAM-deficient cells

• Imaging approaches:

  • Immunofluorescence co-localization of ALCAM and CD6 in tissues

  • Live-cell imaging to track interactions during immune synapse formation

  • Two-photon microscopy for in vivo visualization of cell-cell interactions

• Molecular approaches:

  • Mutagenesis of the CD6-binding domain to identify critical residues

  • Domain swapping experiments between mouse and human ALCAM to assess cross-species functionality

These approaches provide complementary data on the physical and functional aspects of ALCAM-CD6 interactions in mouse models .

How can researchers analyze the role of ALCAM in different mouse disease models beyond food allergy?

Researchers can investigate ALCAM's role in various disease models using:

• Autoimmune disease models:

  • Experimental autoimmune encephalomyelitis (EAE)

  • Collagen-induced arthritis

  • Type 1 diabetes models

  • Analysis of T cell activation, proliferation, and cytokine production

• Cancer models:

  • Investigation of ALCAM's reported upregulation in metastasizing melanoma

  • Analysis of tumor infiltrating lymphocytes and their interaction with ALCAM+ cells

  • Metastasis assays in ALCAM-deficient backgrounds

• Infection models:

  • Bacterial and viral challenge models

  • Assessment of pathogen clearance efficiency

  • Analysis of memory T cell formation

• Transplantation models:

  • Graft versus host disease models

  • Organ transplantation studies

  • Analysis of allorecognition and rejection mechanisms

In each model, comparison between wild-type and ALCAM-deficient mice, combined with selective reconstitution experiments, can elucidate ALCAM's specific contributions to disease pathogenesis or resolution .

How should researchers interpret contradictory findings between ALCAM expression and function in different tissues?

When encountering contradictory findings regarding ALCAM expression and function across different tissues, researchers should consider:

• Tissue-specific factors:

  • Different binding partners may predominate in various tissues (CD6 in immune cells, NgCAM in neural cells)

  • Alternative splicing may generate tissue-specific ALCAM isoforms with distinct functions

  • Post-translational modifications may differ between tissues, affecting function

• Methodological considerations:

  • Different detection methods may have varying sensitivities and specificities

  • Sample preparation techniques can affect protein conformation and epitope accessibility

  • Expression analysis at mRNA versus protein levels may yield discrepancies due to post-transcriptional regulation

• Biological complexity:

  • ALCAM may exist in both membrane-bound and soluble forms with potentially opposing functions

  • Compensatory mechanisms may mask phenotypes in certain tissues or conditions

  • Context-dependent signaling downstream of ALCAM may result in different functional outcomes

A comprehensive approach combining multiple detection methods, functional assays, and careful controls is essential for resolving apparent contradictions .

What statistical approaches are most appropriate for analyzing ALCAM expression data in mouse immunological studies?

For robust statistical analysis of ALCAM expression data in mouse immunological studies:

• For comparing expression levels between groups:

  • Parametric tests (t-test, ANOVA) for normally distributed data

  • Non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) for non-normal distributions

  • Consider paired analyses for before/after comparisons within the same animals

• For correlation analyses:

  • Pearson correlation for linear relationships between ALCAM levels and continuous variables

  • Spearman rank correlation for non-linear or ordinal relationships

  • Multiple regression to account for confounding variables

• For longitudinal data:

  • Repeated measures ANOVA or mixed-effects models

  • Area under the curve analyses for temporal expression patterns

  • Time-to-event analyses for studying outcomes related to ALCAM expression

• Sample size considerations:

  • Power analysis to determine appropriate sample sizes based on expected effect sizes

  • Adjustments for multiple comparisons (e.g., Bonferroni, Benjamini-Hochberg)

  • Careful reporting of both statistical and biological significance

These approaches help ensure reliable interpretation of ALCAM expression data while accounting for biological variability inherent in mouse models .

How can researchers effectively compare ALCAM function between mouse models and human clinical samples?

To effectively compare ALCAM function between mouse models and human clinical samples:

• Focus on conserved domains and functions:

  • The CD6-binding domain is highly conserved and mediates cross-species binding

  • Compare functional outcomes of ALCAM-CD6 interactions rather than just expression levels

• Parallel experimental designs:

  • Use identical detection methods where possible

  • Apply similar stimulation conditions in mouse and human cell cultures

  • Develop comparable readouts for functional assays

• Translational approaches:

  • Confirm mouse findings in human primary cells

  • Compare serum ALCAM levels between mouse models and human patients with similar conditions

  • Validate key findings using humanized mouse models

• Accounting for species differences:

  • Acknowledge differences in immune system organization between mice and humans

  • Consider differential expression patterns across species

  • Interpret results within the context of species-specific biology

Research has demonstrated that serum ALCAM levels are elevated in both OVA-challenged mice and children with food allergy, suggesting conserved mechanisms and supporting the translational relevance of mouse ALCAM studies .

What emerging technologies are advancing our understanding of ALCAM biology in mouse models?

Several cutting-edge technologies are enhancing ALCAM research in mouse models:

• Single-cell approaches:

  • Single-cell RNA sequencing to map cell-specific ALCAM expression patterns

  • Single-cell proteomics to identify co-expressed receptors and signaling molecules

  • Spatial transcriptomics to analyze ALCAM expression in tissue contexts

• Advanced imaging:

  • Super-resolution microscopy to visualize ALCAM distribution on cell membranes

  • Intravital microscopy to observe ALCAM-mediated interactions in live animals

  • Correlative light and electron microscopy to link ALCAM localization with ultrastructural features

• Genetic engineering:

  • CRISPR/Cas9 for precise domain-specific modifications of ALCAM

  • Conditional and inducible knockout systems for temporal control of ALCAM deletion

  • Reporter mice expressing fluorescent ALCAM fusion proteins for real-time visualization

• Systems biology:

  • Network analysis to position ALCAM within broader immunological pathways

  • Multi-omics integration to understand ALCAM regulation and function

  • Mathematical modeling of ALCAM-mediated cell-cell interactions

These technologies promise to reveal new aspects of ALCAM biology beyond what conventional approaches have uncovered .

What are potential therapeutic applications based on ALCAM biology in mouse models?

Mouse model studies of ALCAM suggest several potential therapeutic applications:

• For allergic conditions:

  • Anti-ALCAM antibodies to attenuate Th2 responses in food allergy

  • Soluble ALCAM to compete with membrane-bound ALCAM for CD6 binding

  • Small molecule inhibitors of ALCAM-CD6 interaction

• For autoimmune diseases:

  • Modulation of ALCAM-CD6 interactions to regulate T cell activation

  • Targeted delivery of immunosuppressive agents to ALCAM-expressing cells

  • Combination therapies targeting ALCAM and complementary pathways

• For cancer immunotherapy:

  • Exploitation of ALCAM upregulation on metastatic cells for targeted therapy

  • ALCAM-based chimeric antigen receptors for T cell immunotherapy

  • Disruption of ALCAM-mediated tumor cell migration

• For transplantation:

  • Temporary ALCAM blockade to promote transplant tolerance

  • Ex vivo manipulation of donor tissues to modify ALCAM expression

The attenuated immune responses observed in ALCAM-deficient mice in the context of food allergy provide proof-of-concept for targeting ALCAM in hypersensitivity conditions .

What are the current hypotheses regarding ALCAM's role in neuroimmune interactions in mice?

Current hypotheses about ALCAM's role in neuroimmune interactions include:

• Neural development and function:

  • ALCAM/NgCAM interactions may guide axon pathfinding during development

  • ALCAM homotypic interactions might contribute to synapse formation and stability

  • ALCAM expression in the cerebellum suggests roles in motor coordination

• Neuroimmune crosstalk:

  • ALCAM may facilitate interactions between immune cells and neural tissues

  • During neuroinflammation, ALCAM could regulate immune cell infiltration and activity

  • ALCAM-expressing glial cells might modulate T cell responses in the CNS

• Neuroinflammatory conditions:

  • ALCAM may contribute to pathological immune responses in models of multiple sclerosis

  • Blood-brain barrier interactions might be mediated by ALCAM-expressing endothelial cells

  • Therapeutic targeting of ALCAM could potentially modulate neuroinflammation

These hypotheses represent promising areas for future research, combining neuroscience and immunology approaches to understand ALCAM's multifaceted roles in the nervous system and its interactions with the immune system .

What are the optimal storage and handling conditions for anti-ALCAM antibodies used in mouse studies?

For optimal performance of anti-ALCAM antibodies in mouse studies:

• Storage conditions:

  • Store at 2-8°C for up to 12 months from date of receipt as supplied

  • Do not freeze, as this may compromise antibody functionality

  • Protect PE-conjugated antibodies from light to prevent fluorophore degradation

• Working solution preparation:

  • Dilute only the amount needed for immediate use

  • Use appropriate buffers as recommended by the manufacturer

  • Prepare fresh dilutions for each experiment when possible

• Quality control:

  • Include positive and negative controls in each experiment

  • Periodically verify antibody performance with known ALCAM-expressing samples

  • Monitor for lot-to-lot variations when receiving new antibody batches

• Application-specific considerations:

  • For flow cytometry, optimize antibody concentration to maximize signal-to-noise ratio

  • For Western blot, determine optimal reducing or non-reducing conditions

  • For immunohistochemistry, optimize fixation and antigen retrieval methods

Proper storage and handling ensure consistent, reliable results in ALCAM detection across experiments .

How can researchers optimize isolation of primary mouse cells for ALCAM functional studies?

For optimal isolation of primary mouse cells for ALCAM functional studies:

• Mouse splenocyte isolation:

  • Harvest spleens under aseptic conditions

  • Generate single-cell suspensions by gentle mechanical disruption

  • Remove red blood cells using hypotonic lysis

  • Filter cell suspensions to remove debris and cell clumps

  • Assess viability and adjust cell concentration appropriately

• Activation protocols for enhanced ALCAM expression:

  • For T cells: Combine anti-CD3/CD28 stimulation for 24-48 hours

  • For B cells: Use LPS or anti-IgM plus IL-4 for 24-48 hours

  • For dendritic cells: Apply LPS, CpG, or other TLR ligands

• Cell purification approaches:

  • Magnetic bead-based negative selection for untouched cell populations

  • Flow cytometry-based sorting for highest purity

  • Density gradient separation for specific cell types

• Quality control:

  • Verify cell purity by flow cytometry

  • Assess baseline ALCAM expression levels

  • Check cell viability before functional assays

These optimized protocols ensure the isolation of viable, functional cells with physiologically relevant ALCAM expression for downstream analyses .

What experimental design considerations are essential when comparing wild-type and ALCAM-deficient mice?

When designing experiments comparing wild-type and ALCAM-deficient mice, consider:

• Genetic background controls:

  • Use littermate controls whenever possible

  • Ensure all mice are on the same genetic background

  • Consider backcrossing to establish congenic strains if needed

• Age and sex considerations:

  • Age-match mice precisely due to potential age-related changes in ALCAM expression

  • Include both sexes or justify single-sex use based on preliminary data

  • Control for estrous cycle in female mice if relevant to the research question

• Environmental factors:

  • House wild-type and ALCAM-deficient mice under identical conditions

  • Consider co-housing to normalize microbiome effects

  • Standardize handling, feeding, and other environmental variables

• Experimental protocol design:

  • Blind investigators to genotype during data collection and analysis

  • Randomize treatment assignments

  • Include appropriate positive and negative controls

  • Determine sample size through power analysis

  • Plan for multiple readouts to comprehensively assess phenotype

• Validation approaches:

  • Consider rescue experiments with ALCAM reconstitution

  • Use multiple methods to assess the same parameter

  • Include time course analyses for dynamic processes