CD4L-2 Antibody, FITC conjugated

What is CD4L-2 Antibody, FITC conjugated and what is its target?

CD4L-2 Antibody, FITC conjugated is a polyclonal antibody raised in rabbit against a specific peptide sequence (amino acids 59-77) from the Grass carp CD4-like protein 2 . This antibody specifically recognizes the CD4L-2 protein in Grass carp (Ctenopharyngodon idella, also known as Leuciscus idella) . The antibody has been conjugated with fluorescein isothiocyanate (FITC), a fluorescent dye that enables direct visualization of the target protein in applications such as flow cytometry and fluorescence microscopy. The unique UniProt ID for the target protein is A0A1B2FIB0 .

What are the storage and handling recommendations for CD4L-2 Antibody, FITC conjugated?

The CD4L-2 Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . It is important to avoid repeated freeze-thaw cycles as this can compromise antibody integrity and performance . The antibody is supplied in liquid form in a buffer containing 0.03% Proclin 300 as a preservative, with 50% glycerol and 0.01M PBS at pH 7.4 . These buffer components help maintain antibody stability during storage. For short-term use during experiments, the antibody can be stored at 4°C, but should be returned to -20°C or -80°C for long-term storage to preserve functionality.

What are the recommended applications for CD4L-2 Antibody, FITC conjugated?

Based on available data, CD4L-2 Antibody, FITC conjugated is primarily intended for research applications in Grass carp immunology . While specific validated applications are not extensively documented in the provided literature, the FITC conjugation makes this antibody particularly suitable for:

• Flow cytometry analysis of CD4L-2 expression on carp immune cells

• Fluorescence microscopy for tissue localization studies

• Immunophenotyping of carp lymphocyte populations

These applications align with the typical uses of FITC-conjugated antibodies against CD4 in other species, such as the mouse CD4 antibody (RM4-5 clone) which is well-documented for flow cytometric analysis . Researchers should conduct preliminary validation studies to optimize the antibody for their specific application and experimental conditions.

How should I design a multicolor flow cytometry experiment incorporating CD4L-2 Antibody, FITC conjugated?

When designing a multicolor flow cytometry experiment including CD4L-2 Antibody, FITC conjugated, follow these methodological steps:

• Fluorochrome selection: Since CD4L-2 is FITC-conjugated (emission ~520 nm), select other fluorochromes with minimal spectral overlap, such as PE (emission ~575 nm), PerCP (emission ~675 nm), or APC (emission ~660 nm) .

• Panel design: For a Level Two multicolor analysis (5-8 colors), organize your panel considering fluorochrome brightness and antigen density. FITC is a moderately bright fluorochrome, so it's best suited for antigens with moderate to high expression levels .

• Controls preparation: Include these essential controls:

  • Single-color compensation controls for each fluorochrome

  • Fluorescence Minus One (FMO) controls excluding the CD4L-2-FITC antibody to accurately set gating boundaries

  • Appropriate isotype control (rabbit IgG-FITC) if measuring subtle shifts in fluorescence

• Titration: Optimize the antibody concentration by testing serial dilutions. For FITC-conjugated antibodies similar to the mouse CD4 antibody, concentrations of ≤0.25 μg per test (defined as the amount needed to stain a sample in 100 μL) are typically effective, with cell numbers ranging from 10^5 to 10^8 cells/test .

• Compensation setup: Use single-color compensation beads rather than cells for more accurate compensation, especially when working with multiple fluorochromes .

This methodological approach helps minimize spectral overlap issues while maximizing the information obtained from each experiment.

What are the key considerations for optimization when using CD4L-2 Antibody, FITC conjugated?

Optimizing experiments with CD4L-2 Antibody, FITC conjugated requires careful attention to several parameters:

By systematically optimizing these parameters, researchers can achieve more reliable and reproducible results when using this antibody.

How can I differentiate between specific and non-specific binding when using CD4L-2 Antibody, FITC conjugated?

Differentiating between specific and non-specific binding is crucial for accurate data interpretation. For CD4L-2 Antibody, FITC conjugated, implement the following methodological approaches:

• Use of appropriate controls:

  • Isotype control: A rabbit IgG-FITC conjugate with the same protein concentration and F/P (fluorophore/protein) ratio as the CD4L-2 antibody should be used to establish background fluorescence levels .

  • Blocking experiments: Pre-incubate cells with unconjugated anti-CD4L-2 antibody before adding the FITC-conjugated version to confirm binding specificity.

  • FMO controls: These are particularly valuable in multicolor experiments to accurately set gates and identify spillover effects .

• Pre-absorption validation:

  • Incubate the antibody with its specific peptide immunogen (the 59-77AA peptide from Grass carp CD4-like protein 2) before staining to verify that this eliminates specific binding.

• Cross-reactivity testing:

  • Test the antibody on cell types known to be negative for CD4L-2 expression to confirm absence of binding.

  • If available, use cells from other fish species to verify specificity to Grass carp.

• Signal pattern analysis:

  • Specific binding typically shows a distinct population shift in flow cytometry, while non-specific binding often presents as a more diffuse increase in fluorescence.

  • Compare staining patterns with published literature on CD4-like proteins in fish species.

These methodological approaches help ensure that experimental observations reflect true CD4L-2 expression rather than artifacts.

What are common troubleshooting issues with FITC-conjugated antibodies and their solutions?

When working with CD4L-2 Antibody, FITC conjugated, researchers may encounter several technical challenges. Here are systematic solutions to common issues:

Additionally, it's important to note that FITC-conjugated antibodies may exhibit different binding kinetics compared to APC-conjugated antibodies, with evidence suggesting monovalent binding for FITC conjugates versus divalent binding for larger fluorophores like APC . This characteristic should be considered when interpreting binding data or comparing results across different conjugates.

How does the binding of FITC-conjugated antibodies differ from other fluorophore conjugates?

Research has demonstrated significant differences in binding properties between different fluorophore conjugates of the same antibody. These differences have important implications for experimental design and data interpretation:

• Binding valency differences: A model developed for anti-human CD4 monoclonal antibodies revealed that FITC conjugates tend to exhibit monovalent binding (one antibody binding site engaging with one CD4 receptor), whereas larger fluorophores like APC allow for divalent binding (both antibody binding sites engaging with CD4 receptors) . This phenomenon is attributed to the size and spatial arrangement of the fluorophore affecting the antibody's ability to engage with multiple epitopes simultaneously.

• Equilibrium binding behavior: Studies measuring mean fluorescence intensity (MFI) in flow cytometry have shown different binding curves for FITC versus APC conjugates of the same antibody . These differences reflect altered equilibrium concentrations of bound antibody conjugates to CD4 receptors on cell surfaces.

• Quantitative implications: When performing quantitative flow cytometry experiments, these binding differences may affect calculations of receptor density and binding kinetics. Researchers should be aware that different conjugates of the same antibody clone may yield different quantitative results.

• Steric considerations: The smaller size of FITC (approximately 389 Da) compared to larger fluorophores like APC (approximately 105 kDa) means less steric hindrance during the initial binding event, but may limit bivalent binding due to conformational constraints .

These findings suggest that when comparing data across different fluorophore conjugates or when switching between conjugates in longitudinal studies, researchers should be cautious about direct numerical comparisons of fluorescence intensity or derived metrics.

How can CD4L-2 Antibody, FITC conjugated be used to study immune responses in fish?

CD4L-2 Antibody, FITC conjugated offers valuable opportunities for investigating immune responses in Grass carp and potentially other fish species. Methodological approaches include:

• Lymphocyte subset identification and quantification:

  • Use flow cytometry to identify and enumerate CD4L-2+ lymphocyte populations in different tissues (blood, spleen, head kidney, gill) under normal and pathogen-challenged conditions.

  • Combine with other markers to characterize T helper-like cell subsets in fish, which may have different functional characteristics than mammalian CD4+ T cells.

• Immune response monitoring:

  • Track changes in CD4L-2+ cell populations during infection, vaccination, or environmental stressors.

  • Correlate CD4L-2+ cell frequency with production of specific cytokines or antibodies to establish functional relationships.

• Comparative immunology:

  • Compare CD4L-2 expression patterns with the well-characterized CD4 expression in mammals to elucidate evolutionary conservation and divergence of immune system components.

  • Investigate whether fish CD4L-2+ cells show functional homology to mammalian CD4+ T helper cells.

• Tissue distribution studies:

  • Use immunohistochemistry or fluorescence microscopy to map the distribution of CD4L-2+ cells in different tissue compartments.

  • Identify potential sites of T cell education and immune regulation in fish.

• Functional studies:

  • Combine with cell sorting to isolate CD4L-2+ populations for functional characterization through in vitro assays or transcriptomic analysis.

  • Use CD4L-2 antibody to block potential functional interactions and assess impact on immune responses.

These approaches can significantly advance our understanding of teleost immune system organization and function, which has both fundamental research value and applications in aquaculture health management.

What methodological considerations are important when comparing CD4L-2 Antibody with mammalian CD4 antibodies?

When conducting comparative studies between fish CD4L-2 and mammalian CD4 antibodies, researchers should consider several important methodological aspects:

• Evolutionary and structural differences:

  • While both target CD4-family proteins, fish CD4L-2 and mammalian CD4 have diverged significantly during evolution. Fish often possess multiple CD4-like genes (CD4L-1, CD4L-2) with different domain organizations compared to the four-domain structure of mammalian CD4 .

  • These structural differences affect epitope availability and antibody binding characteristics, necessitating different optimization strategies.

• Experimental conditions adaptation:

  • Temperature considerations: Fish are ectothermic, with optimal physiological temperatures typically lower than mammals. Antibody incubations may require adjustment to 4-15°C rather than the standard 4°C used for mammalian cells.

  • Buffer composition: Fish cells may require specialized buffers that maintain osmolarity appropriate for fish physiology.

• Functional correlation challenges:

  • Unlike well-characterized mammalian CD4 that associates with p56lck and functions as a co-receptor for MHC class II recognition , the signaling pathways and molecular interactions of fish CD4L-2 are less defined.

  • Functional assays should be designed with these differences in mind, avoiding direct functional equivalence assumptions.

• Cross-reactivity assessment:

  • Unlike mammalian CD4 antibodies that show some cross-reactivity between closely related species, fish antibodies often show strict species specificity.

  • When extending studies beyond Grass carp, careful validation is required even for closely related fish species.

• Data interpretation framework:

  • Flow cytometry data from fish samples often show different scatter characteristics and autofluorescence profiles compared to mammalian samples.

  • Gating strategies developed for mammalian CD4+ T cells may require significant adaptation for fish CD4L-2+ cells.

By acknowledging these differences and adapting methodologies accordingly, researchers can avoid misinterpretation when conducting comparative immunology studies between fish and mammals.

How can CD4L-2 Antibody, FITC conjugated be incorporated into studies of fish disease resistance and vaccination responses?

CD4L-2 Antibody, FITC conjugated provides a valuable tool for investigating immune responses relevant to fish health and disease management. Methodological strategies include:

• Vaccine efficacy assessment:

  • Monitor changes in CD4L-2+ lymphocyte populations before and after vaccination using flow cytometry.

  • Correlate CD4L-2+ cell activation (measured by co-expression of activation markers) with protective immunity following challenge.

  • Track the kinetics of CD4L-2+ cell responses at different time points post-vaccination to identify optimal vaccination protocols.

• Pathogen response characterization:

  • Compare CD4L-2+ cell responses during infection with different pathogens (viral, bacterial, parasitic) to identify pathogen-specific immune signatures.

  • Investigate whether particular CD4L-2+ subpopulations correlate with resistance or susceptibility to specific diseases.

  • Use cell sorting of CD4L-2+ cells from infected fish for transcriptomic or functional analysis to identify response mechanisms.

• Genetic selection marker development:

  • Analyze whether specific patterns of CD4L-2 expression correlate with disease resistance traits.

  • Evaluate if CD4L-2+ cell functionality could serve as a biomarker for selecting disease-resistant broodstock in aquaculture breeding programs.

• Immunostimulant evaluation:

  • Assess how dietary immunostimulants or probiotics affect CD4L-2+ cell populations and functionality.

  • Determine whether pre-treatment with immunomodulators enhances CD4L-2+ cell responses to subsequent vaccination or infection.

• Environmental impact studies:

  • Investigate how environmental stressors (temperature changes, hypoxia, pollutants) affect CD4L-2+ cell populations and their ability to respond to pathogens.

  • Use multiparameter flow cytometry incorporating CD4L-2-FITC along with stress markers to correlate environmental factors with immune competence.

These approaches can generate valuable data for developing more effective vaccination strategies, selective breeding programs, and health management protocols in aquaculture settings.

What are the latest methodological advances in using FITC-conjugated antibodies in flow cytometry?

Recent methodological advances have enhanced the utility of FITC-conjugated antibodies like CD4L-2 in flow cytometry applications:

These methodological advances enhance the precision and reproducibility of experiments utilizing CD4L-2 Antibody, FITC conjugated, enabling more sophisticated immunophenotyping studies in fish immunology.

How can I combine CD4L-2 Antibody, FITC conjugated with functional assays to assess T cell responses in fish?

Integrating CD4L-2 Antibody, FITC conjugated with functional assays provides a more comprehensive understanding of fish T cell immunity. Here's a methodological framework:

• Proliferation assays combined with CD4L-2 staining:

  • Label cells with proliferation dyes (e.g., CFSE or CellTrace Violet) prior to stimulation with mitogens or antigens.

  • After the proliferation period, stain with CD4L-2 Antibody, FITC conjugated to identify which T cell subsets are responding.

  • Flow cytometric analysis will reveal proliferation profiles of CD4L-2+ and CD4L-2- populations.

• Intracellular cytokine staining protocols:

  • Stimulate fish lymphocytes with PMA/ionomycin or specific antigens in the presence of protein transport inhibitors.

  • Surface stain with CD4L-2 Antibody, FITC conjugated, then fix and permeabilize cells.

  • Stain intracellularly for cytokines of interest (e.g., IFN-γ, IL-4, IL-17 homologs in fish) using fluorochromes spectrally compatible with FITC.

  • This approach identifies functional polarization of CD4L-2+ cells.

• Cell sorting and functional testing:

  • Use CD4L-2 Antibody, FITC conjugated to sort pure populations of CD4L-2+ cells.

  • Culture sorted cells under various stimulation conditions to assess proliferation, cytokine production, and helper functions.

  • Alternatively, use sorted cells for transcriptomic analysis to identify activated pathways.

• Phosphoflow cytometry:

  • Stimulate cells with relevant ligands, then fix rapidly to preserve phosphorylation states.

  • Permeabilize and stain for both CD4L-2 and phosphorylated signaling proteins (e.g., p-STAT, p-ERK).

  • This identifies signaling pathways activated in CD4L-2+ cells in response to specific stimuli.

• In vivo tracking of immune responses:

  • Isolate lymphocytes from fish at various timepoints after infection or vaccination.

  • Stain with CD4L-2 Antibody, FITC conjugated along with activation markers.

  • Correlate CD4L-2+ cell dynamics with pathogen clearance or antibody production to establish in vivo relevance.

These integrated approaches connect phenotypic identification of CD4L-2+ cells with their functional capabilities, providing deeper insights into fish T cell immunology.

What considerations are important when interpreting CD4L-2 expression data in comparative immunology studies?

When interpreting CD4L-2 expression data in comparative immunology contexts, researchers should consider several critical factors to avoid misinterpretation:

• Evolutionary context of CD4 family molecules:

  • Teleost fish have undergone a whole genome duplication event, resulting in multiple CD4-like genes that may have different functions than mammalian CD4 .

  • CD4L-2 in Grass carp may not be functionally equivalent to CD4 in mammals, despite similar nomenclature.

  • Expression patterns should be interpreted within the evolutionary framework of teleost immune system development.

• Technical considerations in cross-species comparisons:

  • Different antibody conjugates can exhibit variable binding characteristics as demonstrated in anti-human CD4 antibody studies, where FITC conjugates showed monovalent binding while APC conjugates exhibited divalent binding .

  • When comparing data across species using different antibody formats, these binding differences must be accounted for.

  • Standardization using calibration beads with known numbers of fluorophore molecules can help normalize fluorescence intensity data across different systems.

• Microenvironmental factors affecting expression:

  • Temperature dramatically affects immune cell function in ectothermic fish, unlike in mammals with constant body temperature.

  • Seasonal variations in CD4L-2 expression may occur in fish but would not be expected in mammalian CD4 expression.

  • Water quality parameters may influence CD4L-2 expression in ways not relevant to mammalian systems.

• Tissue distribution differences:

  • The anatomical distribution of lymphoid tissues differs significantly between fish and mammals.

  • CD4L-2 expression in fish-specific lymphoid organs (like head kidney) cannot be directly compared to mammalian lymphoid organs.

  • Normalize data to tissue-specific reference points rather than making direct cross-species comparisons.

• Functional correlation limitations:

  • In mammals, CD4 expression strongly correlates with helper T cell function and MHC class II restriction .

  • These correlations may not hold for CD4L-2 in fish, where adaptive immunity has distinct evolutionary features.

  • Functional assays should accompany expression data to establish the actual immune role of CD4L-2+ cells in fish.

By considering these factors, researchers can develop more nuanced interpretations of CD4L-2 expression data that acknowledge both the similarities and differences between fish and mammalian immune systems.