ITGA6 Antibody, FITC conjugated

What is ITGA6 and what are its primary biological functions?

ITGA6, also known as Integrin alpha-6, CD49f, or VLA-6, is a transmembrane glycoprotein that forms heterodimers with beta subunits, primarily forming ITGA6:ITGB1 (α6β1) and ITGA6:ITGB4 (α6β4) complexes. These integrin complexes serve as crucial cell adhesion molecules with significant roles in cell-extracellular matrix interactions.

The ITGA6:ITGB1 complex functions as a receptor for laminin on platelets and is present in oocytes where it participates in sperm-egg fusion processes . Additionally, the ITGA6:ITGB4 complex serves as a laminin receptor in epithelial cells and plays a critical structural role in hemidesmosomes, which are specialized adhesion structures that anchor epithelial cells to the underlying basement membrane .

Recent research has uncovered important signaling functions of ITGA6. The ITGA6:ITGB4 complex binds to neuregulin 1 (NRG1) via its EGF domain, and this interaction is essential for NRG1-ERBB signaling pathways . Furthermore, this integrin complex binds to insulin-like growth factors IGF1 and IGF2, interactions that are critical for their respective signaling cascades, linking cellular adhesion with growth factor signaling networks .

Beyond these functions, differential expression of ITGA6 has been identified as a marker for distinguishing between various cell populations, including atrial and ventricular cardiomyocytes during development . This varying expression pattern makes ITGA6 an important marker for cell identification and isolation in developmental biology and stem cell research.

What does FITC conjugation mean and how does it enhance ITGA6 antibody applications?

FITC (Fluorescein Isothiocyanate Isomer 1) conjugation refers to the chemical linking of the fluorescent dye FITC to an antibody, in this case, anti-ITGA6 antibodies. This conjugation process creates a direct fluorescent label that eliminates the need for secondary detection reagents in various applications, particularly flow cytometry.

The FITC molecule has an excitation maximum at approximately 495 nm and an emission maximum around 520 nm, producing a bright green fluorescence when excited with the appropriate wavelength light. FITC-conjugated ITGA6 antibodies enable direct visualization and quantification of ITGA6 expression on cell surfaces without requiring additional staining steps .

The conjugation process typically involves purification of the antibody by affinity chromatography followed by the controlled reaction with FITC under optimal conditions. For high-quality conjugates, such as the ABIN2661762 antibody, the final solution is freed from unconjugated FITC to minimize background fluorescence and improve signal-to-noise ratios .

FITC conjugation particularly enhances flow cytometry applications by allowing direct single-step staining protocols, reducing processing time and potential variability. The conjugation also enables multiparameter analysis when combined with antibodies labeled with other fluorophores that have minimal spectral overlap with FITC .

How do different ITGA6 antibody clones compare in research applications?

The search results reveal two primary monoclonal antibody clones used for FITC-conjugated ITGA6 detection: clone 450-30A and clone GoH3, each with distinct characteristics and applications.

Clone 450-30A (ABIN2480806, ab30496) is a mouse monoclonal antibody of the IgG1 isotype that reacts specifically with human ITGA6. This clone was developed using alpha 6 beta 4 integrin purified from A431 cells as the immunogen . It has been validated primarily for flow cytometry applications and has been cited in multiple publications, demonstrating its reliability in research settings .

Clone GoH3 (ABIN2661762/ABIN2661763) is a rat monoclonal antibody of the IgG2a kappa isotype with broader species reactivity, recognizing ITGA6 from human, mouse, and numerous other species including baboon, cynomolgus, rhesus monkey, horse, cow, sheep, pig, dog, cat, rabbit, capuchin monkey, chicken, and chimpanzee . This extensive cross-reactivity makes GoH3 particularly valuable for comparative studies across species. The broader applications of this clone include flow cytometry, cytometry by time of flight (CyTOF), and it has been used in research examining differential expression of ITGA6 in cardiomyocytes .

When choosing between these clones, researchers should consider: (1) target species requirements, with 450-30A being human-specific while GoH3 offers multi-species reactivity; (2) compatibility with other antibodies in multi-color panels, considering the isotype and host species to avoid cross-reactivity; and (3) specific application needs, with GoH3 offering validated use in more diverse applications including CyTOF .

How should flow cytometry experiments be designed when using FITC-conjugated ITGA6 antibodies?

Designing effective flow cytometry experiments with FITC-conjugated ITGA6 antibodies requires careful consideration of multiple factors to ensure reliable and interpretable results. The following methodological approach is recommended:

Sample preparation is critical and should begin with creating a single-cell suspension through appropriate tissue dissociation or cell culture harvesting techniques. For adherent cells expressing high levels of integrins like ITGA6, gentle enzymatic dissociation methods that preserve surface epitopes are essential. EDTA-based dissociation may be preferable to trypsin, which can cleave surface proteins .

When setting up the staining protocol, researchers should first optimize antibody concentration through titration experiments. While manufacturer recommendations provide starting points (e.g., 1:11 dilution for GoH3 clone as mentioned in the literature), optimal concentrations may vary by cell type and application . Staining should be performed in buffer containing protein (typically 1-5% serum or BSA) to reduce non-specific binding.

For multicolor panels including FITC-conjugated ITGA6 antibodies, careful panel design is necessary. Since FITC has broad emission spectra with some overlap into other channels, compensation controls must be included. Single-stained controls for each fluorochrome in the panel and an unstained control are essential for accurate compensation during analysis .

Proper controls must include: (1) unstained cells to determine autofluorescence; (2) isotype controls matching the ITGA6 antibody's isotype (IgG1 for 450-30A or IgG2a kappa for GoH3) and conjugated to FITC; (3) fluorescence minus one (FMO) controls; and (4) positive and negative biological controls where ITGA6 expression status is known . For viability assessment, inclusion of propidium iodide (20 μg/mL) or another compatible viability dye is recommended for excluding dead cells, which can give false positive signals .

What are the critical considerations for ITGA6-FITC antibody validation in research studies?

Validating FITC-conjugated ITGA6 antibodies is essential for ensuring experimental reliability. A comprehensive validation strategy should address specificity, sensitivity, reproducibility, and compatibility with experimental conditions.

For specificity validation, researchers should confirm that the antibody recognizes the intended target by testing cells with known ITGA6 expression profiles. Validation can include parallel analysis with alternative antibody clones or detection methods, knockdown/knockout controls, or blocking experiments using recombinant ITGA6 protein. The distinct staining patterns observed in differential expression studies, such as the ITGA6high and ITGA6low populations in cardiomyocytes, can serve as biological validation of specificity .

Sensitivity assessment requires determining the dynamic range of detection and the minimum number of ITGA6 molecules that can be reliably detected. This can be accomplished through calibration with beads containing known quantities of fluorochrome or by comparing with quantitative methods like qPCR. Researchers should establish clear thresholds distinguishing positive from negative populations, particularly important when studying differential expression levels as seen in the cardiomyocyte studies .

Reproducibility validation involves performing repeated measurements under identical conditions to assess consistency. This should include technical replicates (same sample, multiple measurements), biological replicates (different samples from same experimental group), and inter-operator reproducibility if multiple researchers will be conducting the experiments.

For application-specific validation, researchers should confirm compatibility with sample preparation methods, fixation protocols (if required), and staining buffers. Some epitopes may be sensitive to certain fixatives or permeabilization agents. When analyzing tissues or complex cell mixtures, validation should include comparison to established markers for the cell populations of interest and confirmation of expected expression patterns .

How can researchers optimize multicolor panels incorporating ITGA6-FITC antibodies?

Optimizing multicolor flow cytometry panels that include FITC-conjugated ITGA6 antibodies requires strategic planning to ensure minimal spectral overlap, appropriate signal resolution, and compatibility between markers.

When designing the panel, researchers should first consider the brightness of FITC relative to the expression level of ITGA6 in their target cells. FITC has medium brightness compared to other fluorochromes, making it suitable for moderately expressed antigens. For low-expression targets, brighter fluorochromes like PE or APC might be preferable alternatives to FITC-conjugated antibodies, as indicated by the availability of these conjugates in the search results .

Spectral overlap management is crucial for accurate data interpretation. Since FITC has significant emission overlap with PE and other fluorochromes in the yellow-green spectrum, researchers should avoid combining FITC with these fluorochromes on antigens expressed by the same cell populations. When such combinations are unavoidable, careful compensation is essential. The exclusion gating strategy should follow a logical sequence: first excluding doublets and debris, then dead cells using propidium iodide or other viability dyes, followed by blood cells or other contaminating populations before analyzing ITGA6 expression .

For marker compatibility, researchers should consider both the biology of co-expression and technical aspects. When studying cardiomyocytes, for example, combining ITGA6-FITC with markers like alpha-actinin (detected via indirect immunofluorescence) can help correlate ITGA6 expression with differentiation status . Antibody clones should be selected to avoid host species cross-reactivity in the detection system.

Titration of all antibodies in the panel is essential for optimal signal-to-noise ratio. Each antibody, including ITGA6-FITC, should be individually titrated on relevant samples to determine the concentration that gives the maximum separation between positive and negative populations with minimal background. Standard protocols suggest starting with manufacturer recommendations (e.g., 1:11 dilution for certain ITGA6 antibodies) and testing serial dilutions from there .

What strategies are recommended for analyzing differential ITGA6 expression in heterogeneous cell populations?

Analyzing differential ITGA6 expression in heterogeneous cell populations requires sophisticated gating strategies and analytical approaches that can distinguish meaningful biological variation from technical artifacts.

The primary gating strategy should begin with clear population identification. As demonstrated in cardiomyocyte studies, researchers should first exclude doublets, cell debris, blood cells, and dead cells (using propidium iodide at 20 μg/mL) from the analysis . Setting analysis gates should be guided by unstained controls, fluorescence minus one (FMO) controls, or secondary antibody controls to establish accurate positive/negative boundaries .

For analyzing differential expression levels rather than simple positive/negative categorization, researchers should employ histogram analysis or contour plots to visualize the distribution of ITGA6 expression. As seen in the cardiomyocyte research, distinct populations can be identified as ITGA6high and ITGA6low, requiring quantitative thresholds rather than binary gates . The resolution of these populations can be enhanced by adjusting the flow cytometer's PMT voltages and compensation settings to maximize the dynamic range for FITC detection.

Correlation with functional or developmental parameters is critical for meaningful interpretation. In studies examining ITGA6 expression in cardiomyocytes, researchers correlated expression levels with developmental stages and functional characteristics . Similar approaches can be applied to other cell types by correlating ITGA6 expression with proliferation markers like Ki-67, differentiation markers, or functional assays appropriate to the cell type.

Quantitative analysis methods should go beyond simple percentage reporting. Mean or median fluorescence intensity (MFI) values provide information about the quantity of ITGA6 molecules per cell. Calculating the ratio of signal to background or stimulation index can normalize for experiment-to-experiment variation. For more sophisticated analysis, algorithms like t-SNE or UMAP can help visualize and identify populations in high-dimensional data sets that include ITGA6 and multiple other markers.

How do results from ITGA6-FITC antibodies compare across different platforms and detection methods?

Integrating and comparing ITGA6 expression data across different detection platforms and methodologies requires understanding the technical differences between approaches and implementing appropriate normalization strategies.

Flow cytometry with FITC-conjugated ITGA6 antibodies offers single-cell resolution and quantitative assessment of surface expression levels. The sensitivity depends on the antibody clone, fluorochrome brightness, and instrument settings. From the search results, we see that clones like GoH3 and 450-30A have been validated for flow cytometry applications, with FITC serving as a medium-brightness fluorochrome suitable for detecting moderate to high expression levels .

Comparing flow cytometry with immunohistochemistry (IHC) or immunofluorescence (IF) requires consideration of the different information provided by each method. While flow cytometry quantifies ITGA6 expression in dissociated cells, IHC and IF preserve spatial information and can reveal localization patterns within tissues or subcellular compartments. The search results indicate that several ITGA6 antibody clones are compatible with multiple applications including IHC, IF, and flow cytometry, though not all are available with FITC conjugation for every application .

For cross-platform normalization, researchers should implement standardization approaches. When comparing flow cytometry data across instruments or time points, calibration beads with known quantities of fluorochrome should be used to standardize fluorescence intensity measurements. For comparing flow cytometry with protein quantification methods like Western blot, correlation studies may be necessary to establish equivalency relationships.

Technical variations between platforms can significantly impact results. For example, the epitope recognized by an antibody may be differently accessible in flow cytometry versus fixed tissues in IHC. Different fixation and permeabilization protocols can affect staining intensity and pattern. When comparing results across platforms, researchers should use the same antibody clone when possible and validate the correlation between methods using reference samples with known expression levels.

What are the key considerations when interpreting ITGA6 expression in relation to developmental and disease states?

Interpreting ITGA6 expression data in developmental and disease contexts requires integration of quantitative measurements with biological understanding of ITGA6's functional roles in different cellular processes.

In developmental biology, ITGA6 expression dynamics can serve as key indicators of cellular differentiation and specialization. The search results highlight how differential ITGA6 expression characterizes atrial versus ventricular cardiomyocytes throughout development . When interpreting such patterns, researchers should consider that ITGA6 expression changes may precede morphological or functional changes, making it a valuable early marker for developmental transitions.

The heterodimeric partners of ITGA6 significantly influence its biological function and should be considered in data interpretation. ITGA6 primarily pairs with either β1 (ITGB1) or β4 (ITGB4) integrin subunits, forming receptors with distinct signaling properties and cellular roles . The ITGA6:ITGB1 complex functions in laminin binding on platelets and in sperm-egg fusion, while ITGA6:ITGB4 serves as a critical component of hemidesmosomes in epithelial cells . Comprehensive interpretation of ITGA6 expression should include assessment of these binding partners when possible.

In disease contexts, particularly cancer research, ITGA6 expression has been associated with stem cell-like properties, invasion, and metastasis in multiple tumor types. Interpreting ITGA6 expression in such settings requires correlation with clinical parameters, other molecular markers, and functional assays. Changes in glycosylation or conformational states of ITGA6 that may not be detected by all antibody clones can have significant functional implications, so using multiple detection methods or antibody clones may provide more complete information.

For quantitative interpretation, establishing meaningful thresholds is essential. In the cardiomyocyte studies, researchers distinguished ITGA6high from ITGA6low populations and correlated these with functional parameters . Similar approaches can be applied to other systems, but thresholds should be determined based on biological relevance rather than arbitrary divisions, potentially through correlation with functional assays or other established markers.

How can ITGA6-FITC antibodies be utilized for isolation and characterization of stem cell populations?

FITC-conjugated ITGA6 antibodies have become powerful tools for isolating and characterizing stem cell populations across various tissue types, leveraging ITGA6's role as a marker for stem and progenitor cells.

For cell isolation protocols, fluorescence-activated cell sorting (FACS) using ITGA6-FITC antibodies allows precise separation of cell populations based on expression levels. The choice of antibody clone is critical - GoH3 clone has broad species cross-reactivity (human, mouse, and other species), making it versatile for comparative studies across model organisms . The sorting protocol should be optimized to maintain cell viability, typically using buffer containing protein supplements and maintaining cells at 4°C during processing to minimize integrin internalization. Gate settings should be established using appropriate controls as described in cardiomyocyte studies, with unstained, FMO, and secondary antibody controls guiding threshold determination .

Post-isolation characterization is essential for confirming stemness properties. The protocol described in the search results demonstrates how sorted ITGA6high and ITGA6low cardiomyocyte populations can be further characterized by immunofluorescence staining for markers like alpha-actinin and Ki-67 . This approach allows correlation of ITGA6 expression with both differentiation status (alpha-actinin) and proliferative capacity (Ki-67), providing functional validation of the isolated populations. Quantification through manual counting of Ki-67+/DAPI+ nuclei ratios offers a standardized method for assessing proliferation potential between different ITGA6-expressing fractions .

Molecular profiling of isolated populations can provide deeper insights into the biological significance of differential ITGA6 expression. RNA sequencing, proteomics, or targeted gene expression analysis of sorted ITGA6high versus ITGA6low populations can reveal associated pathways and molecular signatures. Such analyses have been particularly valuable in understanding the role of ITGA6 in maintaining stem cell properties in various tissues and in cancer stem cells.

Functional validation through in vitro and in vivo assays is the ultimate test of stemness properties in ITGA6-defined populations. Researchers typically assess self-renewal through serial passaging or colony-forming assays, differentiation potential through directed differentiation protocols, and in vivo regenerative capacity through transplantation studies. The correlation between ITGA6 expression levels and these functional properties can establish its utility as a stem cell marker in specific tissue contexts.

What methodologies are recommended for studying ITGA6 in complex tissue microenvironments?

Studying ITGA6 in complex tissue microenvironments requires specialized approaches that preserve spatial relationships while enabling quantitative assessment of expression and function.

Multiparameter imaging techniques offer powerful tools for examining ITGA6 expression in tissue context. Multiplex immunofluorescence combining FITC-conjugated ITGA6 antibodies with markers for other cell types, extracellular matrix components, and signaling molecules can reveal complex interaction networks. When designing such panels, spectral overlap must be carefully managed, potentially using linear unmixing algorithms for closely overlapping fluorophores. For applications requiring maximum spatial resolution, super-resolution microscopy techniques can be employed to examine the nanoscale organization of ITGA6 in structures like hemidesmosomes.

Single-cell analysis approaches bridge flow cytometry and imaging techniques. Single-cell RNA sequencing of dissociated tissues followed by computational reconstruction of spatial relationships can reveal how ITGA6 expression correlates with cell state and position within tissues. Mass cytometry (CyTOF) using metal-tagged antibodies against ITGA6 and other markers provides highly multiplexed data with single-cell resolution. The search results indicate that clone GoH3 has been validated for CyTOF applications, making it suitable for such advanced analytical approaches .

For functional studies in tissue context, organoid models provide a valuable middle ground between 2D culture and in vivo systems. ITGA6 expression and function can be studied in organoids derived from primary tissues or stem cells, with the advantage of preserving 3D architecture and cell-cell interactions while allowing experimental manipulation. Live imaging of fluorescently labeled ITGA6 in organoid cultures can reveal dynamic aspects of expression and localization during developmental processes or in response to stimuli.

In situ hybridization techniques complement protein-level analyses by revealing ITGA6 mRNA expression patterns. Fluorescence in situ hybridization (FISH) can be combined with immunofluorescence to correlate mRNA and protein expression at the single-cell level in tissue sections. This approach is particularly valuable for understanding transcriptional regulation of ITGA6 in different microenvironmental contexts.

How can researchers investigate the functional interplay between ITGA6 and its binding partners?

Investigating the functional interactions between ITGA6 and its binding partners requires specialized methodological approaches that go beyond simple expression analysis to examine protein-protein interactions, conformational states, and downstream signaling events.

Co-immunoprecipitation studies represent a foundational approach for validating direct interactions between ITGA6 and partner proteins. While FITC-conjugated antibodies are primarily designed for flow cytometry, unconjugated antibodies of the same clone can be used for immunoprecipitation, as indicated by the IP capabilities of the GoH3 clone mentioned in the search results . Sequential immunoprecipitation with antibodies against ITGA6 followed by immunoblotting for suspected binding partners (ITGB1, ITGB4, NRG1, IGF1, IGF2) can confirm these interactions in cellular contexts of interest.

Proximity ligation assays (PLA) offer an elegant approach for visualizing protein-protein interactions in situ. This technique uses pairs of antibodies against ITGA6 and a potential binding partner, followed by a ligation reaction that generates a fluorescent signal only when the proteins are in close proximity (<40 nm). PLA can reveal not only whether interactions occur but also their subcellular localization and response to stimuli or inhibitors.

For investigating signaling dynamics, phospho-specific antibodies against downstream effectors can be combined with ITGA6-FITC in flow cytometry or imaging applications. This approach can reveal how ITGA6 engagement triggers signaling cascades and how these signals vary between cell populations with different ITGA6 expression levels. The search results highlight important signaling roles for ITGA6:ITGB4 complexes in NRG1-ERBB and IGF1/IGF2 pathways that could be investigated using such approaches .

Functional blocking studies provide direct evidence of ITGA6's role in specific cellular processes. Function-blocking antibodies against ITGA6 (potentially the same clones used for detection but in unconjugated form) can interrupt interactions with laminin or other binding partners. Comparing the effects of such blocking on ITGA6high versus ITGA6low populations, as defined by flow cytometry with FITC-conjugated antibodies, can reveal differential dependence on ITGA6 signaling.

CRISPR-Cas9 genetic manipulation combined with ITGA6-FITC flow cytometry enables sophisticated structure-function studies. Introducing mutations in ITGA6 or its binding partners, followed by analysis of complex formation, localization, and downstream signaling, can dissect the molecular determinants of these interactions. The differential expression of ITGA6 in cardiomyocyte populations provides a valuable model system for such studies, allowing comparison of genetic manipulations in distinct cellular contexts .