ITGAL Monoclonal Antibody,FITC Conjugated

What is ITGAL and what is its significance in immunological research?

ITGAL (Integrin Alpha-L), also known as CD11a, is the alpha subunit of lymphocyte function-associated antigen-1 (LFA-1), a 170-180 kDa type I transmembrane glycoprotein. It forms a heterodimeric integrin with the beta 2 chain (ITGB2/CD18), creating a receptor for intercellular adhesion molecules (ICAMs 1-3). ITGAL plays critical roles in leukocyte adhesion, migration, and immune cell interactions. It is expressed on multiple leukocyte populations, including B and T lymphocytes, monocytes, macrophages, neutrophils, basophils, and eosinophils, making it an important marker in immunological research . Research interest in ITGAL stems from its involvement in various immune phenomena, including leukocyte-endothelial cell interactions, cytotoxic T-cell mediated killing, and antibody-dependent killing by granulocytes and monocytes .

What are the differences between human and mouse ITGAL antibodies?

Researchers must carefully select species-specific ITGAL antibodies for their experiments:

While these antibodies target homologous proteins, they are not interchangeable due to species-specific epitope differences. Researchers must validate reactivity for their specific experimental system .

How should FITC-conjugated ITGAL antibodies be stored and handled?

Proper storage and handling of FITC-conjugated ITGAL antibodies are critical for maintaining their performance:

FITC-conjugated antibodies are light-sensitive and require specific storage conditions. Store these antibodies in the dark at 4°C to prevent photobleaching of the fluorochrome. Do not freeze FITC-conjugated antibodies as this can damage both the protein and fluorochrome . Most preparations are supplied in PBS buffer (pH 7.4) containing 0.09% sodium azide, which acts as a preservative. Care should be taken when handling, as sodium azide is a hazardous substance that should be managed only by trained personnel .

For optimal performance, avoid repeated exposure to light during experimental procedures. Prepare working aliquots to minimize freeze-thaw cycles of the stock solution. Following these storage guidelines will help maintain antibody performance and fluorescence intensity over time .

What basic applications are suitable for FITC-conjugated ITGAL antibodies?

FITC-conjugated ITGAL antibodies are primarily optimized for certain research applications:

Flow cytometry represents the primary application for these antibodies, with manufacturers specifically recommending them for this purpose . The recommended working concentration for flow cytometry is typically around 3 μg/mL, though optimal concentrations should be determined experimentally for each specific application . Some antibody clones, like 4A122, are also suitable for immunofluorescence microscopy applications .

When using these antibodies, researchers should include appropriate controls, such as isotype controls (IgG1 or IgG2a depending on the antibody clone) and fluorescence-minus-one (FMO) controls to establish proper gating strategies and identify true positive populations .

What are optimal panel design considerations when using FITC-conjugated ITGAL antibodies?

When designing multicolor flow cytometry panels including FITC-conjugated ITGAL antibodies, researchers should consider several critical factors:

FITC has significant spectral overlap with other fluorochromes, particularly PE, which necessitates proper compensation. ITGAL is typically highly expressed on leukocytes, making FITC a suitable fluorochrome choice despite its moderate brightness compared to newer alternatives. For complex panels (>8 colors), consider the brightness hierarchy - FITC is of medium brightness and may be better suited for higher-expressed antigens like ITGAL .

Panel design should account for the expression level of ITGAL on target populations. The antibody concentration should be titrated to determine optimal signal-to-noise ratio, typically starting with the manufacturer's recommended concentration (e.g., 3 μg/mL) and testing serial dilutions . When performing dual or multi-color analysis, pair FITC with fluorochromes having minimal spectral overlap, such as APC or PE-Cy7, to minimize compensation requirements.

How can researchers troubleshoot weak or inconsistent FITC-ITGAL signal?

When encountering weak or inconsistent ITGAL-FITC signals, researchers should systematically evaluate these technical parameters:

Systematic evaluation of these parameters can help identify the source of technical issues. Additionally, running known positive controls (such as peripheral blood leukocytes) can help distinguish between technical and biological variables affecting ITGAL detection .

How do different fixation methods affect FITC-ITGAL antibody performance?

Fixation and permeabilization can significantly impact FITC-ITGAL antibody staining and results:

For protocols requiring both surface ITGAL detection and intracellular staining, it's recommended to perform surface staining with FITC-ITGAL antibody prior to fixation and permeabilization steps. When fixation cannot be avoided before staining, researchers should validate that their fixation protocol doesn't interfere with detection of their specific ITGAL epitope by comparing fixed and unfixed samples .

How can FITC-conjugated ITGAL antibodies be used to study leukocyte activation?

FITC-conjugated ITGAL antibodies offer valuable insights into leukocyte activation states:

ITGAL undergoes conformational changes upon leukocyte activation, potentially affecting epitope accessibility. By monitoring ITGAL expression levels and distribution patterns using FITC-conjugated antibodies, researchers can track activation-dependent changes. ITGAL clustering at the immunological synapse during T cell activation can be visualized using sub-saturating concentrations of non-blocking FITC-ITGAL antibody clones .

For quantitative assessment, researchers should examine both the percentage of ITGAL-positive cells and the median fluorescence intensity (MFI), as activation may alter expression levels or surface distribution rather than the percentage of positive cells. Combining ITGAL-FITC with markers of cellular activation (CD25, CD69, HLA-DR) provides a more comprehensive analysis of the activation state . ITGAL expression and distribution can serve as indicators of functional changes in leukocyte adhesion, migration, and effector functions .

What methodological approaches can distinguish between different conformational states of ITGAL?

Distinguishing between inactive and active ITGAL conformations requires specialized approaches:

ITGAL/LFA-1 exists in multiple conformational states that affect its binding to ICAMs. Standard FITC-conjugated antibodies typically detect total ITGAL levels regardless of conformation . To specifically assess active conformations, researchers can use activation conditions such as PMA, Mg²⁺/EGTA, or specific activating antibodies before staining with FITC-ITGAL antibodies.

Combining standard ITGAL-FITC antibodies with conformation-specific antibodies (in different fluorochromes) allows simultaneous assessment of total and active ITGAL populations. Correlating ITGAL staining patterns with functional assays, such as adhesion to ICAM1-coated surfaces, provides insight into the relationship between detected ITGAL and its functional state . When interpreting changes in ITGAL staining, researchers should consider whether alterations represent actual expression changes versus conformational changes affecting epitope accessibility .

How can FITC-conjugated ITGAL antibodies be combined with other markers for immunophenotyping?

Effective combination of FITC-ITGAL antibodies with other markers enables comprehensive leukocyte subpopulation analysis:

When designing multicolor panels, consider the relative expression levels of each marker and select appropriate fluorochromes. FITC works well for highly expressed markers like ITGAL . For complex immunophenotyping, use hierarchical gating strategies starting with lineage markers before analyzing ITGAL expression on specific subpopulations .

Modern analysis approaches like dimensionality reduction (t-SNE, UMAP) can help identify patterns of ITGAL expression across multiple cellular populations simultaneously, potentially revealing novel associations between ITGAL expression and specific cell states or functions .

How can FITC-conjugated ITGAL antibodies be used to investigate the immunological synapse?

FITC-conjugated ITGAL antibodies provide valuable tools for studying immunological synapse formation:

The immunological synapse represents a specialized cell-cell junction where ITGAL/LFA-1 plays a critical role in adhesion and signaling. ITGAL acts as a platform at the immunological synapse to translate TCR engagement and density of ICAM1 into graded adhesion responses . Using non-blocking FITC-ITGAL antibody clones at sub-saturating concentrations allows visualization of ITGAL redistribution during synapse formation without interfering with function.

For optimal results, researchers should combine ITGAL-FITC staining with markers of TCR engagement and co-stimulatory molecules. Advanced imaging techniques such as TIRF or confocal microscopy enable detailed visualization of ITGAL distribution within the synapse structure. Quantitative image analysis can measure ITGAL clustering and co-localization with other synapse components . Time-lapse imaging with FITC-ITGAL antibodies can capture the dynamics of ITGAL redistribution during synapse formation and maturation.

What sample preparation techniques optimize ITGAL detection across different tissue types?

Sample preparation significantly impacts ITGAL detection with FITC-conjugated antibodies:

For optimal results, process tissues rapidly and maintain cold temperatures (4°C) during preparation to preserve ITGAL expression and minimize internalization. Use enzyme inhibitors when enzymatic tissue digestion is necessary. For tissues with high autofluorescence in the FITC channel, consider alternative fluorochromes or implement autofluorescence reduction strategies .

When working with fixed tissues, validate that fixation doesn't interfere with your specific ITGAL epitope recognition. For frozen tissue sections, acetone fixation may better preserve ITGAL epitopes compared to paraformaldehyde .

How do different FITC-conjugated ITGAL antibody clones compare in research applications?

A comparative analysis of commonly used FITC-conjugated ITGAL antibody clones reveals important differences:

When selecting an antibody clone, researchers should consider not only species reactivity but also the specific experimental application. Different clones may exhibit varying sensitivity for detecting ITGAL in different contexts. Validation experiments comparing multiple clones can help identify the optimal antibody for specific research questions .

Some clones may partially block ITGAL-ICAM interactions, which should be considered when designing functional studies. The immunogen source (e.g., MEM-25 from leukemia patient leukocytes, M17/4 from mouse splenic T cells) may affect epitope specificity and performance in different applications .

How does ITGAL function in different immune contexts and how can this be studied?

ITGAL plays diverse roles across various immune contexts that can be investigated using FITC-conjugated antibodies:

ITGAL/LFA-1 functions as a receptor for multiple ICAMs (ICAM1, ICAM2, ICAM3, ICAM4) and other binding partners including F11R and the secreted form of ISG15 . It contributes significantly to natural killer cell cytotoxicity and is involved in leukocyte adhesion and transmigration . ITGAL also plays a critical role in lymphoid development, with research showing it's required for generation of common lymphoid progenitor cells in bone marrow .

Using FITC-conjugated ITGAL antibodies, researchers can track expression changes during immune responses, correlating with functional outcomes. Flow cytometric analysis combined with functional assays (migration, adhesion, cytotoxicity) provides insight into how ITGAL expression relates to functional capacity. Blocking studies using unconjugated antibodies of the same clone can help establish ITGAL's role in specific immune functions, with FITC-conjugated antibodies later used to quantify expression .

What are the critical factors to consider when interpreting changes in ITGAL expression?

When interpreting changes in ITGAL expression using FITC-conjugated antibodies, researchers should consider both technical and biological factors:

Technical factors include potential photobleaching of FITC, requiring standardized acquisition timing across samples. ITGAL internalization following activation may reduce detectable surface signal, potentially leading to misinterpretation of expression changes. Epitope masking through ligand binding or conformational changes may affect antibody accessibility to certain epitopes .

Biological considerations include distinguishing between actual protein expression changes and conformational changes affecting epitope accessibility. Microenvironmental influences such as pH and calcium levels can affect both ITGAL conformation and FITC fluorescence. Integrin clustering may occur without changes in total expression, requiring careful interpretation of distribution patterns .

For robust analysis, researchers should compare both percentage of positive cells and median fluorescence intensity (MFI). Consider using active conformation-specific antibodies alongside total ITGAL antibodies for comprehensive assessment. Time-course experiments can help distinguish between transient and sustained changes in ITGAL expression or conformation .

How can FITC-conjugated ITGAL antibodies be used in clinical research applications?

FITC-conjugated ITGAL antibodies have valuable applications in clinical research contexts:

In autoimmune and inflammatory disease research, ITGAL expression on specific leukocyte populations may correlate with disease activity or treatment response. Standardized flow cytometry protocols using FITC-ITGAL antibodies can facilitate multi-center clinical studies by providing consistent measurement of this marker across patient samples .

For transplantation research, monitoring ITGAL expression on recipient lymphocytes may provide insights into allograft rejection processes. In cancer immunology, analyzing ITGAL on tumor-infiltrating lymphocytes can help assess their migratory and functional potential .

When developing clinical research protocols, researchers should establish standardized sample processing and staining procedures to minimize technical variability. Calibration beads should be used to standardize fluorescence measurements across different instruments and time points. Appropriate reference ranges should be established for specific patient populations and cellular subsets .