ICAM3 Antibody, FITC conjugated

What is ICAM3 and why is it significant in immunological research?

ICAM3 (CD50) is an adhesion molecule belonging to the immunoglobulin gene superfamily that functions primarily as a ligand for β2 integrins including LFA-1, Mac-1, and αdβ2 . Its expression is restricted to cells of the hematopoietic lineage, making it a valuable marker for studying leukocyte biology . The significance of ICAM3 in immunological research stems from its role in cell-cell interactions, particularly in the initial contact between dendritic cells and T cells that supports primary immune responses . Additionally, ICAM3 shows regulated expression during processes such as monocyte-to-macrophage differentiation and dendritic cell maturation, suggesting its importance in immune cell development and function .

How does a FITC-conjugated ICAM3 antibody differ from other conjugates, and when should it be preferentially used?

FITC-conjugated ICAM3 antibodies utilize fluorescein isothiocyanate as the fluorochrome, which emits green fluorescence (approximately 519 nm) when excited with blue light (approximately 495 nm) . Compared to other conjugates like PE (phycoerythrin) or biotin, FITC offers several distinct characteristics. FITC has a relatively high quantum yield, making it suitable for detecting medium to high-abundance antigens such as ICAM3 on leukocytes . FITC-conjugated antibodies should be preferentially used when:

• Performing multicolor flow cytometry with non-overlapping fluorochromes

• Working with samples that have minimal autofluorescence in the green spectrum

• Conducting experiments where photobleaching is not a significant concern

• Analyzing samples where quick, direct detection without amplification steps is desired

The choice of FITC over other conjugates should be based on the specific experimental design, instrument configuration, and the need for compatibility with other fluorochromes in multiparameter analyses .

What are the key structural and functional domains of ICAM3 that influence antibody binding?

ICAM3 contains distinct functional domains that influence antibody binding efficacy and specificity. Based on the available research, antibodies targeting different amino acid regions of ICAM3 demonstrate varying experimental utilities. For instance, antibodies targeting the AA 200-547 region of ICAM3 have been developed for various applications including flow cytometry . This region encompasses several immunoglobulin-like domains that are critical for the protein's adhesion functions.

The interaction between ICAM3 and its binding partners involves specific structural elements:

• N-terminal domains (approximately AA 30-203) that contribute to LFA-1 binding and are targeted by monoclonal antibodies like MEM-04 for functional studies

• Central regions (approximately AA 200-547) that maintain the protein's tertiary structure and are recognized by polyclonal antibodies used in multiple applications including ELISA and flow cytometry

• C-terminal domains involved in intracellular signaling pathways

The binding specificity of anti-ICAM3 antibodies is influenced by the conformation of these domains, and researchers should select antibodies targeting appropriate epitopes based on whether they need to block functional interactions or simply detect ICAM3 presence .

How can FITC-conjugated ICAM3 antibodies be optimally used in flow cytometry experiments?

For optimal use of FITC-conjugated ICAM3 antibodies in flow cytometry, researchers should implement the following methodological approaches:

• Sample preparation: Freshly isolated cells yield better results than frozen samples. For leukocytes, standard protocols using PBS with 1-2% FBS or BSA as staining buffer work effectively .

• Titration: Determine the optimal antibody concentration through titration experiments. While manufacturers may suggest starting dilutions, cell-specific expression levels of ICAM3 vary significantly between cell types, necessitating optimization .

• Compensation controls: Due to FITC's spectral overlap with other fluorophores like PE, proper compensation controls are essential. Single-stained controls should be prepared using the same cells as the experimental samples .

• Live/dead discrimination: Include viability dyes compatible with FITC (avoiding those with similar emission spectra) to exclude dead cells, which can bind antibodies non-specifically .

• Analysis parameters: Gate strategies should account for the dynamic expression of ICAM3, particularly when studying processes like monocyte differentiation where ICAM3 levels change significantly .

• Storage considerations: FITC conjugates are more sensitive to photobleaching than other fluorophores, so samples should be protected from light and analyzed promptly after staining .

When analyzing monocyte-derived cells, researchers should be aware that ICAM3 expression diminishes upon monocyte-to-macrophage differentiation, which can be a useful marker for differentiation status when combined with other lineage markers such as CD163 .

What protocols are recommended for fixation when using FITC-conjugated ICAM3 antibodies in immunofluorescence microscopy?

When using FITC-conjugated ICAM3 antibodies for immunofluorescence microscopy, fixation protocols must balance preserving ICAM3 epitope structure with maintaining cell morphology and FITC fluorescence intensity. Based on research protocols, the following approaches are recommended:

Researchers should note that ICAM3 distribution patterns change significantly during cellular processes like monocyte migration and dendritic cell maturation, so fixation timing is crucial when studying dynamic processes .

How do you design multicolor panels incorporating FITC-conjugated ICAM3 antibodies to study hematopoietic cell differentiation?

Designing effective multicolor panels incorporating FITC-conjugated ICAM3 antibodies requires strategic selection of complementary markers and fluorophores. For studying hematopoietic cell differentiation, the following approach is recommended:

• Panel design principles:

  • Place FITC (ICAM3) in a channel with high signal-to-noise ratio since ICAM3 expression changes during differentiation

  • Avoid fluorophores with spectral overlap with FITC (PE-CF594, PerCP) for critical markers

  • Reserve brighter fluorophores (PE, APC) for markers with lower expression levels

• Marker selection for monocyte-to-macrophage differentiation:

  Marker	Purpose	Recommended Fluorophore
  ICAM3 (CD50)	Differentiation status	FITC
  CD14	Monocyte/macrophage identity	APC or BV421
  CD163	M2 polarization	PE
  HLA-DR	Activation status	BV605
  CD80/CD86	M1 polarization	PE-Cy7

• Controls and validation:

  • Include FMO (Fluorescence Minus One) controls for accurate gating

  • Validate panel using known differentiation models (e.g., GM-CSF vs. M-CSF induced differentiation)

  • Track ICAM3 expression kinetics alongside established differentiation markers

• Analysis strategy:

  • Implement hierarchical gating to first identify viable cells and relevant populations

  • Use bivariate plots of ICAM3 versus differentiation markers to track population transitions

  • Apply dimensionality reduction algorithms (tSNE, UMAP) for complex differentiation landscapes

Research shows that ICAM3 expression decreases during monocyte-to-macrophage differentiation, making it a useful marker when tracked alongside differentiation-specific markers like CD163 . This approach enables identification of transitional cell states based on the gradual loss of ICAM3 expression coupled with the acquisition of macrophage-specific markers.

How does ICAM3 expression change during immune cell differentiation and activation, and how can FITC-conjugated antibodies track these changes?

ICAM3 expression undergoes significant changes during immune cell differentiation and activation, making FITC-conjugated antibodies valuable tools for tracking cellular transitions. Research has demonstrated several key patterns:

• Monocyte-to-macrophage differentiation:

  • ICAM3 expression progressively decreases during differentiation

  • This downregulation is observed in both M1 (GM-CSF-induced) and M2 (M-CSF-induced) macrophage polarization pathways

  • qRT-PCR analysis shows corresponding reduction in ICAM3 mRNA levels, indicating transcriptional regulation

• Dendritic cell maturation:

  • Immature dendritic cells express higher levels of ICAM3

  • Upon maturation triggered by inflammatory stimuli, ICAM3 expression decreases

  • This coincides with increased expression of RUNX3, suggesting a potential regulatory mechanism

• Transendothelial migration:

  • Monocytes display reduced ICAM3 expression after migrating through endothelial barriers

  • This change occurs rapidly during the migration process and precedes differentiation changes

To effectively track these changes using FITC-conjugated ICAM3 antibodies, researchers should:

• Establish baseline expression levels in relevant progenitor populations

• Implement time-course experiments with consistent staining protocols

• Use appropriate statistical methods to quantify gradual expression changes

• Combine with functional assays to correlate ICAM3 expression with specific cellular functions

Flow cytometric analysis using FITC-conjugated antibodies allows for precise quantification of these expression changes, particularly when mean fluorescence intensity (MFI) values are tracked alongside the percentage of positive cells .

What is the relationship between RUNX transcription factors and ICAM3 expression, and how might this impact experimental design?

The relationship between RUNX transcription factors and ICAM3 expression represents a sophisticated regulatory mechanism that researchers should consider when designing experiments involving ICAM3. Evidence demonstrates that RUNX transcription factors negatively regulate ICAM3 expression through several mechanisms:

• Direct promoter regulation:

  • RUNX binding sites have been identified in the ICAM3 gene promoter region

  • EMSA experiments confirm that RUNX1 and RUNX3 directly bind to these regulatory elements

  • Disruption of RUNX-binding sites enhances promoter activity, confirming a repressive function

• Inverse correlation during differentiation:

  • ICAM3 levels diminish during monocyte-derived macrophage differentiation

  • This coincides with increased RUNX3 expression in the differentiated cells

  • siRNA-mediated reduction of RUNX3 results in increased ICAM3 mRNA levels, confirming the regulatory relationship

• Cooperative regulation with other transcription factors:

  • RUNX factors collaborate with Ets-1 and C/EBPα in ICAM3 transactivation

  • Mutation of RUNX binding elements abrogates this collaborative effect

  • This suggests complex regulatory networks controlling ICAM3 expression

These findings impact experimental design in several ways:

• When studying ICAM3 expression changes, researchers should consider monitoring RUNX3 levels as a potential mechanism

• For genetic manipulation experiments, targeting RUNX factors can provide a method to modulate ICAM3 expression

• In transcriptional studies, the ICAM3 promoter can serve as a model for investigating repressive transcriptional mechanisms

• When analyzing differentiation processes, the RUNX3-ICAM3 axis serves as a molecular signature of cellular transitions

Understanding this regulatory relationship provides deeper insight into the biological significance of ICAM3 expression patterns observed in flow cytometry experiments using FITC-conjugated antibodies .

How do ICAM3-Fc fusion proteins compare with anti-ICAM3 antibodies in targeting and functional studies?

ICAM3-Fc fusion proteins and anti-ICAM3 antibodies exhibit distinct properties in targeting and functional studies that researchers should consider when designing experiments:

These comparative insights suggest that while FITC-conjugated anti-ICAM3 antibodies excel in detection and quantification applications, ICAM3-Fc fusion proteins may offer superior functionality in certain immunomodulatory applications. Researchers should select the appropriate tool based on whether their experimental goals prioritize detection sensitivity or functional potency .

What are common challenges in flow cytometry when using FITC-conjugated ICAM3 antibodies, and how can they be addressed?

Researchers frequently encounter specific challenges when using FITC-conjugated ICAM3 antibodies in flow cytometry. Here are the most common issues and their methodological solutions:

• Autofluorescence interference:

  • Problem: Certain cell types (particularly macrophages and dendritic cells) exhibit significant autofluorescence in the FITC channel

  • Solution: Implement an autofluorescence control (unstained cells) and consider using spectral unmixing algorithms. Alternatively, select a different fluorophore like Alexa Fluor 488 which offers better signal-to-noise ratio while still being detected in the FITC channel

• Photobleaching during processing:

  • Problem: FITC is particularly susceptible to photobleaching during sample processing

  • Solution: Minimize exposure to light, perform staining in amber tubes, add samples to the flow cytometer immediately before acquisition, and include a FITC-single stained control immediately before and after the experimental run to monitor signal degradation

• Signal intensity variations during differentiation studies:

  • Problem: ICAM3 expression decreases during monocyte differentiation, potentially dropping below detection thresholds

  • Solution: Use antibody clones with higher affinity (like MEM-171), optimize PMT voltages to detect low expression levels, and include positive controls with known high ICAM3 expression to confirm staining efficacy

• Epitope masking in certain conditions:

  • Problem: Cellular activation or differentiation can alter ICAM3 conformation, potentially masking epitopes

  • Solution: Compare results using antibodies targeting different ICAM3 domains (e.g., AA 30-203 vs. AA 200-547) to ensure comprehensive detection

• Non-specific binding:

  • Problem: FITC-conjugated antibodies can sometimes exhibit non-specific binding to Fc receptors on myeloid cells

  • Solution: Include an Fc receptor blocking step (using human IgG or commercial Fc block) in the staining protocol, and validate results with isotype controls conjugated to FITC

By implementing these methodological approaches, researchers can significantly improve data quality and reliability when using FITC-conjugated ICAM3 antibodies in flow cytometry experiments, particularly in differentiation studies where expression levels change dynamically .

How should results be interpreted when ICAM3 expression patterns contradict other differentiation markers?

When ICAM3 expression patterns contradict other differentiation markers, researchers should implement a structured analytical approach to resolve these discrepancies:

• Hierarchical marker assessment:

  • Establish a priority framework based on the lineage specificity and stability of each marker

  • Consider that ICAM3 regulation is influenced by multiple factors beyond differentiation, including RUNX transcription factors and inflammatory signals

  • Weigh the relative significance of contradictory markers against established differentiation paradigms

• Temporal dynamics analysis:

  • Implement time-course experiments to determine whether contradictions represent transitional states

  • ICAM3 downregulation during monocyte-to-macrophage differentiation follows specific kinetics that may not precisely align with other markers

  • Asynchronous regulation of different markers is biologically significant and should be documented rather than dismissed as experimental error

• Microenvironmental influence assessment:

  • Evaluate whether culture conditions (cytokines, serum factors, cell density) differentially affect ICAM3 versus other markers

  • Document whether contradictory patterns emerge in specific subpopulations, suggesting heterogeneous responses

  • Consider whether cell-cell contact influences ICAM3 expression independently of differentiation state

• Methodological validation:

  • Confirm findings using alternative detection methods (qRT-PCR for mRNA levels, western blot)

  • Verify antibody performance using positive and negative controls under identical conditions

  • Consider whether differences in epitope accessibility might explain contradictory results

• Biological interpretation framework:

  Observation Pattern	Potential Interpretation	Validation Approach
  ICAM3 downregulation without other differentiation markers	Early response to environmental signals preceding differentiation	Time-course correlation with RUNX3 expression
  Persistent ICAM3 expression despite differentiation markers	Subset-specific regulation or activation state	Single-cell analysis combining ICAM3 with functional assays
  Heterogeneous ICAM3 in homogeneous populations	Cell cycle-dependent regulation or asymmetric division	Cell cycle marker correlation

Rather than dismissing contradictory results, researchers should recognize that such discrepancies often reveal important biological insights about the regulation of cellular differentiation processes and the specific roles of ICAM3 in immune cell biology .

What controls are essential for validating FITC-conjugated ICAM3 antibody specificity in experimental systems?

Validating the specificity of FITC-conjugated ICAM3 antibodies requires a comprehensive set of controls to ensure accurate interpretation of experimental results. The following controls are essential:

• Isotype controls:

  • Primary control: FITC-conjugated isotype-matched control antibody (e.g., FITC-conjugated rabbit IgG for polyclonal antibodies or mouse IgG1 for monoclonal antibodies like MEM-171)

  • Purpose: Establishes baseline fluorescence and identifies non-specific binding through Fc receptors

  • Implementation: Apply at the same concentration as the ICAM3 antibody under identical conditions

• Biological negative controls:

  • Cells known to lack ICAM3 expression (non-hematopoietic lineage cells)

  • ICAM3-knockout or ICAM3-silenced cells (if available)

  • Fully differentiated macrophages with confirmed low ICAM3 expression

  • Purpose: Confirms absence of non-specific binding to other cellular components

• Biological positive controls:

  • Cell lines with stable, high ICAM3 expression (THP-1, K-562, Jurkat)

  • Freshly isolated peripheral blood monocytes

  • Purpose: Validates antibody binding to the target under optimal conditions

• Epitope competition controls:

  • Pre-incubation with unlabeled anti-ICAM3 antibody targeting the same epitope

  • Purpose: Confirms specificity through signal reduction when binding sites are blocked

  • Implementation: Titration of unlabeled antibody should produce dose-dependent reduction in FITC signal

• Method validation controls:

  • Correlation with alternative detection methods (Western blot, qRT-PCR for ICAM3 mRNA)

  • Cross-validation with antibodies targeting different ICAM3 epitopes

  • Purpose: Confirms that the observed signal represents actual ICAM3 expression rather than technical artifacts

• Functional validation:

  • ICAM3 blocking experiments to confirm functional relevance of detected protein

  • Correlation with known biological responses (e.g., changes during differentiation)

  • Purpose: Links the detected molecule to expected biological functions

Implementation of this comprehensive control strategy ensures that experimental observations can be confidently attributed to specific ICAM3 detection, particularly in complex experimental systems involving cellular differentiation or activation where expression patterns change dynamically .

How are FITC-conjugated ICAM3 antibodies being used to study novel aspects of immune cell function?

FITC-conjugated ICAM3 antibodies are enabling novel investigations into immune cell function across several cutting-edge research areas:

• Immune cell heterogeneity mapping:

  • High-dimensional flow cytometry incorporating ICAM3-FITC with other markers allows identification of previously uncharacterized immune cell subsets

  • ICAM3 expression patterns are being used as predictive markers for functional responses to stimuli, revealing functional heterogeneity within phenotypically similar populations

  • This approach has revealed that differential ICAM3 expression correlates with distinct migratory and antigen-presenting capacities among monocyte subpopulations

• Extracellular vesicle (EV) characterization:

  • FITC-conjugated ICAM3 antibodies are being applied to analyze EVs released during immune cell activation and differentiation

  • Flow cytometric analysis of ICAM3+ EVs provides insights into intercellular communication mechanisms

  • The presence of ICAM3 on EVs may influence their targeting to specific recipient cells, representing a novel mechanism of immune regulation

• Immune cell migration dynamics:

  • Real-time imaging using FITC-conjugated ICAM3 antibodies enables visualization of receptor redistribution during migration

  • This approach has revealed that ICAM3 undergoes specific redistribution patterns during transendothelial migration of monocytes

  • The dynamic changes in ICAM3 localization correlate with migration efficiency and subsequent differentiation potential

• Transcriptional regulation networks:

  • Combined flow cytometry with ICAM3-FITC and intracellular transcription factor staining provides insights into the relationship between RUNX3 expression and ICAM3 regulation

  • This approach has confirmed the inverse relationship between RUNX3 and ICAM3 at the single-cell level during differentiation processes

  • The methodology reveals how transcriptional networks dynamically regulate adhesion molecule expression during immune cell development

These emerging applications demonstrate how FITC-conjugated ICAM3 antibodies contribute to our understanding of complex immune cell behaviors beyond their traditional use in phenotypic identification, particularly in contexts where dynamic changes in expression patterns provide functional insights .

What are the current limitations in ICAM3 research, and how might they be addressed with new methodological approaches?

Current ICAM3 research faces several significant limitations that emerging methodological approaches aim to address:

• Temporal resolution limitations:

  • Current challenge: Most studies provide static snapshots of ICAM3 expression rather than dynamic temporal profiles

  • Methodological solution: Implementation of time-lapse flow cytometry or continuous imaging using photoconversion-tagged ICAM3 antibodies to track expression changes in living cells with high temporal resolution

  • Potential impact: Would reveal the kinetics of ICAM3 regulation during immune cell activation and differentiation, providing mechanistic insights

• Cellular heterogeneity masking:

  • Current challenge: Bulk analysis methods obscure cell-to-cell variations in ICAM3 expression and function

  • Methodological solution: Integration of FITC-conjugated ICAM3 antibodies into single-cell RNA sequencing workflows through CITE-seq approaches

  • Potential impact: Would correlate ICAM3 protein expression with comprehensive transcriptional profiles at single-cell resolution

• Limited understanding of regulatory mechanisms:

  • Current challenge: While RUNX transcription factors are known to regulate ICAM3, the complete regulatory network remains poorly characterized

  • Methodological solution: CUT&RUN or CUT&Tag approaches combined with ICAM3 expression analysis to identify additional transcription factors binding to the ICAM3 promoter

  • Potential impact: Would elucidate the full range of transcriptional regulators controlling ICAM3 expression in different cellular contexts

• Functional significance ambiguity:

  • Current challenge: The physiological consequences of altered ICAM3 expression during differentiation remain incompletely understood

  • Methodological solution: Development of conditional ICAM3 knockout models or CRISPR-based approaches to modulate ICAM3 expression at specific differentiation stages

  • Potential impact: Would establish causal relationships between ICAM3 expression changes and functional outcomes during immune cell differentiation

• Technical limitations in detection sensitivity:

  • Current challenge: FITC photobleaching and autofluorescence can limit detection of low ICAM3 expression

  • Methodological solution: Implementation of brighter fluorophores (Brilliant Violet™ dyes) or signal amplification approaches (tyramide signal amplification)

  • Potential impact: Would enable detection of ICAM3 on rare cell populations or at low expression levels during terminal differentiation stages

By addressing these limitations through innovative methodological approaches, researchers can advance our understanding of ICAM3 biology beyond current constraints, particularly regarding its dynamic regulation and functional significance in immune cell development and function .

How might ICAM3-targeting approaches using FITC-conjugated antibodies contribute to therapeutic development?

While primarily research tools, FITC-conjugated ICAM3 antibodies are contributing to therapeutic development strategies through several innovative approaches:

• Targeted nanoparticle delivery systems:

  • Current research demonstrates that ICAM3-Fc coated nanoparticles efficiently deliver antigens to dendritic cells, enhancing cross-presentation and CD8+ T cell activation

  • FITC-conjugated ICAM3 antibodies enable rapid screening and optimization of targeting efficiency in preclinical development

  • Flow cytometric analysis using these antibodies helps determine whether therapeutic nanoparticles might compete with endogenous ICAM3 for receptor binding

• Immunomodulatory therapeutic discovery:

  • FITC-conjugated ICAM3 antibodies facilitate high-throughput screening of compounds that modulate ICAM3 expression

  • Compounds identified through such screens could potentially regulate immune cell differentiation or function through ICAM3-dependent mechanisms

  • The finding that RUNX transcription factors negatively regulate ICAM3 provides potential targets for therapeutic intervention in conditions where modulating ICAM3 expression would be beneficial

• Cell therapy manufacturing optimization:

  • ICAM3 expression dynamics during monocyte-to-macrophage differentiation serve as quality control markers in cell therapy production

  • FITC-conjugated antibodies enable real-time monitoring of these changes during manufacturing processes

  • Optimizing culture conditions based on ICAM3 expression patterns may improve the functional properties of cell therapy products

• Biomarker development:

  • Changes in ICAM3 expression during immune cell differentiation and activation provide potential biomarkers for disease states characterized by altered myeloid cell function

  • FITC-conjugated antibodies enable standardized detection methods suitable for clinical laboratory implementation

  • Correlation of ICAM3 expression patterns with disease progression or treatment response could inform therapeutic decision-making

• Mechanistic understanding for targeted therapies:

  • Research using FITC-conjugated ICAM3 antibodies has revealed that the ICAM3-DC-SIGN interaction contributes to T cell activation mechanisms

  • This mechanistic insight guides the development of therapies targeting this pathway for immunomodulation

  • The finding that ICAM3-Fc outperforms antibodies in certain functional contexts suggests novel approaches to therapeutic design

These translational applications demonstrate how FITC-conjugated ICAM3 antibodies contribute to therapeutic development beyond their conventional use as research reagents, particularly in understanding the mechanistic basis for potential interventions targeting ICAM3-dependent processes .