CD58 Antibody, FITC

What is CD58 and why is it important in immunological research?

CD58, also known as lymphocyte function-associated antigen-3 (LFA-3), is a 60-70 kDa glycoprotein member of the immunoglobulin superfamily . It exists in two forms: a transmembrane form with a cytoplasmic domain and a glycosylphosphatidylinositol (GPI)-linked form . CD58 has significant importance in immunological research because it functions as a ligand for the T-lymphocyte CD2 glycoprotein . This interaction is crucial for:

• Mediating thymocyte interactions with thymic epithelial cells

• Facilitating antigen-independent and antigen-dependent interactions of T-lymphocytes with target cells and antigen-presenting cells

• Enhancing T-lymphocyte rosetting with erythrocytes

• Priming responses in both CD2+ and CD58+ cells

The CD58-CD2 interaction plays a fundamental role in T cell activation pathways, making it an essential target for studying immune responses in both normal and pathological conditions.

What are the primary applications of CD58-FITC antibodies in research?

CD58-FITC antibodies are primarily utilized in flow cytometry applications . They enable researchers to:

• Quantify CD58 expression on cell surfaces

• Analyze CD58+ cell populations in mixed samples

• Study changes in CD58 expression during immune responses

• Investigate CD58's role in pathological conditions

These antibodies have proven particularly valuable in:

• Flow cytometric analysis of human peripheral blood cells

• Detection of minimal residual disease in acute lymphocytic leukemia

• Evaluation of liver damage related to hepatitis B

• Investigation of T cell activation mechanisms

For optimal results, the antibodies are typically used at a concentration of 20 μl per 10^6 cells for direct immunofluorescence staining, though researchers should titrate reagents for their specific applications .

What sample preparation protocols are recommended for CD58-FITC antibody staining?

For effective CD58-FITC antibody staining, the following protocol is generally recommended:

• Collect blood samples using EDTA as an anticoagulant

• Transfer 100 μl of anticoagulated blood to a 12 × 75 mm polystyrene test tube (containing approximately 10^6 cells)

• Add 20 μl of CD58-FITC antibody (this volume is a guideline; optimal volume should be determined by each laboratory)

• Mix gently with a vortex mixer

• Incubate in the dark at room temperature (time typically specified in product instructions)

• Wash twice with PBS

• Centrifuge at 2500 r/min and discard supernatant

• Add 1 ml of PBS-formaldehyde to fix cells

• Analyze using a flow cytometer

This procedure ensures optimal staining while preserving cellular integrity for accurate analysis of CD58 expression patterns.

What controls should be included when working with CD58-FITC antibodies?

When conducting experiments with CD58-FITC antibodies, proper controls are essential for accurate interpretation of results. Researchers should include:

• Isotype control: A FITC-conjugated IgG1 antibody (matching the CD58 antibody's isotype) to assess non-specific binding

• Negative cell population control: Cell types known not to express CD58 or samples from healthy individuals to establish baseline measurements

• Positive control: Samples known to express CD58 at high levels to confirm antibody functionality

• Unstained control: Cells processed without antibody to assess autofluorescence

• Single-stained controls: When performing multi-parameter analysis, include single-stained samples for proper compensation setup

These controls help distinguish specific from non-specific signals and ensure reliable quantification of CD58 expression across experimental conditions.

How do membrane-bound and soluble CD58 levels correlate with disease progression in hepatitis B?

Research has revealed important correlations between CD58 expression and hepatitis B pathogenesis. Both soluble CD58 (sCD58) in serum and membrane CD58 on peripheral blood mononuclear cells (PBMCs) show significant elevation in hepatitis B patients compared to healthy controls .

A key study demonstrated that CD58 levels directly correlate with liver damage severity in hepatitis B patients. Specifically:

• Levels of sCD58 in serum and membrane CD58 in PBMCs were significantly higher in hepatitis B patients than in normal controls (p < 0.05)

• CD58 levels (both serum and membrane-bound) showed positive correlation with liver function markers:

  • Total bilirubin (TBIL)

  • Direct bilirubin (DBIL)

  • Indirect bilirubin (IBIL)

  • Alanine aminotransferase (ALT)

  • Aspartate aminotransferase (AST)

These findings suggest that CD58 monitoring using CD58-FITC antibodies could serve as a valuable biomarker for assessing liver damage severity in hepatitis B infection. The correlation between CD58 levels and conventional liver function tests indicates its potential utility in monitoring disease progression and treatment response.

What methodological considerations are important for multi-parameter flow cytometry using CD58-FITC alongside other markers?

When incorporating CD58-FITC antibodies into multi-parameter flow cytometry panels, several methodological considerations are crucial:

• Spectral overlap management:

  • FITC emission spectrum overlaps with PE and other fluorochromes

  • Proper compensation setup is essential using single-stained controls

  • Consider alternative fluorochromes for markers with similar expression patterns to CD58

• Panel design optimization:

  • Place CD58-FITC on a different parameter than markers with potential co-expression

  • Account for relative expression levels (bright markers on dimmer fluorochromes)

  • Consider using CD58 antibodies conjugated to alternative fluorochromes if expression is expected to be low

• Titration of all antibodies:

  • The recommended volume of 20 μl per 10^6 cells is a starting point

  • Each laboratory should determine optimal concentration to maximize signal-to-noise ratio

• Sample processing consistency:

  • Standardize procedures for sample collection, storage, and preparation

  • Maintain consistent incubation times and temperatures

  • Process all experimental groups simultaneously when possible

• Data analysis strategy:

  • Define clear gating strategies incorporating CD58 expression patterns

  • Consider computational approaches for high-dimensional data analysis

  • Include appropriate visualization methods for complex relationships

By addressing these considerations, researchers can obtain reliable multi-parameter data incorporating CD58 expression alongside other immunological markers.

How can CD58-FITC antibodies be utilized for studying the CD58-CD2 interaction in T cell activation models?

CD58-FITC antibodies provide valuable tools for investigating the CD58-CD2 interaction in T cell activation models through several methodological approaches:

• Co-culture systems analysis:

  • Label antigen-presenting cells (APCs) with CD58-FITC antibodies

  • Quantify CD58 expression on APCs before and during interaction with T cells

  • Track changes in CD58 distribution during immunological synapse formation

  • Correlate CD58 expression levels with functional outcomes (cytokine production, proliferation)

• Blocking studies:

  • Use non-conjugated CD58 antibodies to block CD58-CD2 interactions

  • Compare functional outcomes with CD58-FITC staining intensity

  • Assess dose-dependent effects of blocking on T cell activation parameters

• Signaling pathway investigation:

  • CD58 triggers protein kinase activity differently between its transmembrane and GPI-anchored forms

  • The GPI-anchored form is constitutively associated with protein kinases

  • The transmembrane form activates kinase activity upon triggering

  • Researchers can use CD58-FITC to identify and sort cell populations expressing different CD58 isoforms for further signaling studies

• Functional correlation analysis:

  • CD58-CD2 interaction enhances:

    • Interferon γ and IL-2 mRNA transcription and translation

    • CD4+ T cell differentiation toward Th1 phenotype

    • Initiation of cell-mediated immune responses

  • Quantitative analysis of CD58 expression using CD58-FITC can be correlated with these functional outcomes

These methodological approaches enable researchers to dissect the complex roles of CD58 in T cell activation, providing insights into basic immunological mechanisms and potential therapeutic targets.

What are the technical considerations for using CD58-FITC antibodies in minimal residual disease detection?

CD58-FITC antibodies serve as powerful tools for detecting minimal residual disease (MRD) in acute lymphocytic leukemia . To optimize their use in this context, researchers should consider:

• Sensitivity optimization:

  • Use high-quality, bright FITC conjugates with optimal fluorochrome-to-protein ratios

  • Employ cytometers with appropriate laser and detector configurations

  • Acquire sufficient events (typically >500,000) to detect rare populations

  • Consider signal amplification techniques for very low-level expression

• Specificity enhancement:

  • Combine CD58-FITC with other leukemia-associated immunophenotypic markers

  • Develop detailed gating strategies based on normal expression patterns

  • Include appropriate negative controls from healthy individuals

  • Validate findings with molecular techniques when possible

• Standardization protocols:

  • Establish consistent sample processing procedures

  • Standardize instrument settings across time points

  • Use fluorescent beads for day-to-day calibration

  • Participate in external quality assessment programs

• Data analysis strategies:

  • Employ computational approaches for identifying aberrant populations

  • Consider machine learning algorithms for pattern recognition

  • Establish clear positivity thresholds based on control populations

  • Correlate findings with clinical outcomes to establish prognostic value

• Sample considerations:

  • Process samples within 24 hours of collection when possible

  • Standardize fixation protocols to preserve cellular integrity

  • Consider cryopreservation effects on CD58 expression if samples cannot be processed immediately

By addressing these technical considerations, researchers can maximize the utility of CD58-FITC antibodies for MRD detection, potentially improving patient stratification and treatment decisions.

How do different sample preparation techniques affect CD58-FITC antibody staining results?

Sample preparation methodology significantly impacts CD58-FITC antibody staining outcomes. Researchers should consider:

• Anticoagulant selection:

  • EDTA is commonly recommended for blood collection

  • Heparin may activate cells and alter surface expression patterns

  • Citrate may impact calcium-dependent processes

• Timing considerations:

  • Process samples within 24 hours of collection for optimal results

  • Extended storage may lead to decreased antigen expression

  • Document time between collection and staining for protocol standardization

• Cell isolation methods:

  • Density gradient separation may selectively lose certain cell populations

  • Direct staining of whole blood preserves physiological conditions but introduces complexity

  • PBMC isolation through centrifugation is commonly used for CD58 analysis

  • Each method may yield different baseline expression levels

• Fixation protocols:

  • PBS-formaldehyde is commonly used for fixation

  • Paraformaldehyde concentration (typically 1-4%) affects signal intensity

  • Fixation duration impacts epitope preservation

  • Some epitopes may be sensitive to specific fixatives

• Permeabilization considerations:

  • Not typically required for CD58 detection as it's a surface molecule

  • If intracellular pools require examination, detergent selection is critical

  • Saponin, Triton X-100, and methanol have different effects on epitope preservation

For optimal protocol development, researchers should conduct comparative studies with their specific samples and CD58-FITC antibody clones to determine which preparation techniques yield the most consistent and biologically relevant results.

What approaches can be used to quantify changes in CD58 expression during immune responses?

Accurate quantification of CD58 expression changes during immune responses requires systematic methodological approaches:

• Quantitative flow cytometry methods:

  • Mean/median fluorescence intensity (MFI) measurement

  • Molecules of equivalent soluble fluorochrome (MESF) calibration

  • Antibody binding capacity (ABC) determination using standardized beads

  • Consider the non-linear relationship between fluorescence and antibody binding

• Temporal analysis strategies:

  • Establish clear baseline measurements before stimulation

  • Define appropriate time points based on expected kinetics

  • Maintain consistent processing times for all samples

  • Consider both short-term (minutes to hours) and long-term (days) changes

• Stimulation protocols:

  • Document precise stimulation conditions (concentrations, durations)

  • Include relevant controls for each stimulation condition

  • Consider physiologically relevant stimuli for the research question

  • Compare multiple stimulation methods when appropriate

• Complementary techniques:

  • Flow cytometry using CD58-FITC for per-cell analysis

  • ELISA for soluble CD58 quantification in supernatants/serum

  • qPCR for transcriptional regulation analysis

  • Western blotting for total protein assessment

  • Consider integrating multiple approaches for comprehensive analysis

• Data normalization approaches:

  • Internal controls (unchanging markers)

  • Ratio to baseline expression

  • Percent change calculations

  • Statistical analysis appropriate for the data distribution

Using these methodological approaches, researchers can generate reliable quantitative data on CD58 expression dynamics during various immune responses, contributing to a deeper understanding of its biological roles.

How can researchers troubleshoot common issues with CD58-FITC antibody staining?

When working with CD58-FITC antibodies, researchers may encounter several technical challenges. Here are methodological approaches to troubleshoot common issues:

• Weak or absent signal:

  • Verify antibody viability (check expiration date, storage conditions)

  • Increase antibody concentration (titrate to determine optimal amount)

  • Extend incubation time (typically 20-30 minutes at room temperature)

  • Check flow cytometer laser alignment and detector settings

  • Verify sample viability (>90% viable cells recommended)

  • Consider alternative clones if epitope accessibility is an issue

• High background/non-specific staining:

  • Include proper blocking steps (serum matching secondary host)

  • Reduce antibody concentration through titration

  • Ensure thorough washing between steps

  • Use appropriate isotype controls to determine background levels

  • Check for potential cross-reactivity with other species/proteins

  • Consider autofluorescence quenching techniques

• Inconsistent results between experiments:

  • Standardize all protocol steps (timing, temperatures, reagent lots)

  • Use calibration beads to normalize instrument settings

  • Process all experimental groups simultaneously when possible

  • Implement quality control procedures for antibody performance

  • Document all procedural details for reproducibility

• Unexpected expression patterns:

  • Confirm antibody specificity using known positive and negative controls

  • Compare with literature reports for expected expression patterns

  • Consider biological variables (activation status, disease state)

  • Verify using alternative detection methods or antibody clones

  • Examine potential modulation of CD58 expression by experimental conditions

• Poor resolution between positive and negative populations:

  • Optimize cytometer settings (PMT voltage, compensation)

  • Consider alternative fluorochromes with higher quantum yield

  • Implement appropriate gating strategies based on controls

  • Examine potential downregulation or shedding of CD58 under experimental conditions

By systematically addressing these issues using the suggested approaches, researchers can optimize CD58-FITC antibody staining protocols for their specific experimental systems.

What are the current research findings regarding CD58 expression in disease states?

Research utilizing CD58-FITC antibodies has revealed significant insights into CD58 expression patterns across various disease states:

• Hepatitis B infection:

  • Both soluble CD58 (sCD58) in serum and membrane CD58 in PBMCs are significantly elevated in patients compared to healthy controls

  • CD58 levels positively correlate with markers of liver damage (TBIL, DBIL, IBIL, ALT, AST)

  • This suggests CD58 is closely associated with hepatitis B pathogenesis and liver damage severity

• Acute lymphocytic leukemia:

  • CD58 serves as a powerful marker for detecting minimal residual disease

  • Aberrant CD58 expression patterns help distinguish leukemic blasts from normal progenitors

  • CD58-FITC antibodies enable sensitive detection of residual disease populations

• T cell-mediated immunity:

  • CD58-CD2 interactions play crucial roles in:

    • Antigen-specific T cell activation enhancement

    • Cell-mediated cytotoxicity mechanisms

    • T lymphocyte adhesion to target cells

  • These findings highlight CD58's importance in normal immune function and potential dysregulation in disease

• Immunological synapse formation:

  • CD58 participates in immunological synapse formation between T cells and antigen-presenting cells

  • Both transmembrane and GPI-anchored forms contribute differently to signaling

  • The GPI-anchored form is constitutively associated with protein kinases

  • The transmembrane form activates kinase activity upon triggering

These research findings underscore CD58's multifaceted roles in immune regulation and disease processes, highlighting the value of CD58-FITC antibodies as important investigative tools.

How can CD58-FITC antibodies be integrated into comprehensive immunophenotyping panels?

Effective integration of CD58-FITC antibodies into comprehensive immunophenotyping panels requires strategic planning:

• Panel design considerations:

  • Assess CD58 expression levels on target populations to determine appropriate parameter placement

  • Consider spectral overlap with other fluorochromes in the panel

  • Balance bright markers with dimmer fluorochromes

  • Include markers that help define relevant cell subsets where CD58 expression is of interest

• Recommended marker combinations:

  • For T cell interaction studies:

    • CD58-FITC, CD2-PE, CD3-APC, CD4-PerCP, CD8-Pacific Blue

    • This combination allows examination of both sides of the CD58-CD2 interaction

  • For leukemia research:

    • CD58-FITC, CD10-PE, CD19-APC, CD34-PerCP, CD45-V450

    • This panel helps identify abnormal B-cell precursors with aberrant CD58 expression

  • For hepatitis B studies:

    • CD58-FITC, CD3-PE, CD14-APC, CD16-PE-Cy7, HLA-DR-BV421

    • Enables correlation of CD58 expression with immune cell activation status

• Analysis strategies:

  • Implement hierarchical gating approaches, starting with viability and lineage markers

  • Create bivariate plots examining CD58 expression relative to other functional markers

  • Consider dimension reduction techniques (tSNE, UMAP) for high-parameter data visualization

  • Correlate CD58 expression with functional readouts (cytokine production, proliferation)

• Data integration approaches:

  • Combine surface CD58 analysis with soluble CD58 measurements when relevant

  • Correlate CD58 expression with functional assays (cytotoxicity, proliferation)

  • Consider integrating genomic or transcriptomic data when available

  • Implement computational approaches for pattern recognition across multiple parameters

By following these strategies, researchers can effectively incorporate CD58-FITC antibodies into comprehensive immunophenotyping panels, generating multidimensional data that provides deeper insights into CD58's biological roles.

What are the differences between available CD58-FITC antibody clones and their applications?

Several CD58-FITC antibody clones are available for research, each with distinct characteristics and optimal applications:

When selecting between these clones, researchers should consider:

• Epitope accessibility:

  • Different clones recognize distinct epitopes that may be differentially accessible depending on sample preparation

  • Some epitopes may be sensitive to fixation or affected by protein-protein interactions

• Cross-reactivity:

  • While most clones are human-specific, some (like MEM-63) also react with pig samples

  • Always verify species reactivity for your specific research model

• Application compatibility:

  • Match clone selection to intended applications (some perform better in certain techniques)

  • Consider the fluorochrome-to-protein ratio, which may vary between manufacturers

• Validation evidence:

  • Review literature citations for each clone to ensure suitability for your research question

  • Consider performing comparative studies if working with novel cell types or conditions

Understanding these differences enables researchers to select the optimal CD58-FITC antibody clone for their specific experimental requirements.

How does the physiological role of CD58 inform experimental design for immune cell interaction studies?

Understanding CD58's physiological roles provides crucial context for designing meaningful experiments investigating immune cell interactions:

• CD58-CD2 interaction dynamics:

  • CD58 serves as the ligand for CD2 on T lymphocytes

  • This interaction is fundamental for:

    • Thymocyte interactions with thymic epithelial cells

    • Antigen-independent and -dependent T cell interactions with target cells

    • T lymphocyte rosetting with erythrocytes

  • Experimental designs should account for both sides of this interaction

• Cell type considerations:

  • CD58 has wide tissue distribution on:

    • Hematopoietic cells (monocytes, lymphocytes, erythrocytes)

    • Non-hematopoietic cells (endothelial cells, fibroblasts)

  • Different cell types express different ratios of transmembrane vs. GPI-linked forms

  • Erythrocytes exclusively express the GPI-linked form

  • Experimental designs should account for cell type-specific expression patterns

• Functional outcome measurements:

  • CD58-CD2 binding enhances:

    • Antigen-specific T cell activation

    • Cell-mediated cytotoxicity

    • Interferon γ and IL-2 mRNA transcription

    • CD4+ T cell differentiation toward Th1 phenotype

  • Experiments should incorporate readouts for these functional outcomes

• Technical approach selection:

  • For adhesion studies: quantify cell-cell contacts or binding force measurements

  • For activation studies: measure calcium flux, cytokine production, proliferation

  • For signaling studies: examine kinase activation patterns (different between CD58 isoforms)

  • For in vivo relevance: correlate with disease markers (as seen in hepatitis B studies)

By incorporating these physiological insights into experimental design, researchers can develop more robust and biologically relevant protocols for investigating CD58's roles in immune cell interactions.

What future research directions are emerging for CD58-FITC antibody applications?

Several promising research directions are emerging for CD58-FITC antibody applications, presenting opportunities for novel insights:

• Integration with advanced cytometry technologies:

  • Spectral cytometry for enhanced multiplexing capabilities

  • Mass cytometry (CyTOF) using metal-conjugated CD58 antibodies for high-parameter analysis

  • Imaging flow cytometry to examine CD58 localization during immune synapse formation

  • Single-cell sequencing paired with index sorting based on CD58 expression

• Exploration in additional disease contexts:

  • Beyond established applications in hepatitis B and leukemia

  • Investigation of CD58's role in autoimmune disorders

  • Examination of CD58 expression in various cancer microenvironments

  • Potential diagnostic applications in infectious diseases beyond hepatitis B

• Therapeutic monitoring applications:

  • Tracking CD58 expression changes during immunotherapy

  • Monitoring CD58-CD2 interactions following experimental interventions

  • Correlating CD58 levels with treatment response in hepatitis B and other conditions

• Methodological innovations:

  • Development of standardized protocols for CD58 quantification across laboratories

  • Creation of reference materials for absolute quantification

  • Novel computational approaches for analyzing CD58 expression in high-dimensional datasets

  • Integration of artificial intelligence for pattern recognition in complex immunophenotyping data

• Mechanistic studies:

  • Further characterization of differential signaling between CD58 isoforms

  • Investigation of CD58's roles beyond CD2 binding

  • Examination of regulatory mechanisms controlling CD58 expression

  • Exploration of CD58's potential interactions with emerging immunomodulatory pathways

These emerging directions highlight the continuing importance of CD58-FITC antibodies as valuable tools for advancing our understanding of immune regulation and disease processes.

How can researchers optimize data analysis strategies for CD58-FITC-based experiments?

Optimizing data analysis for CD58-FITC-based experiments requires thoughtful implementation of several strategies:

• Standardization approaches:

  • Implement consistent gating strategies across experiments

  • Use fluorescent calibration beads to normalize fluorescence intensity

  • Convert arbitrary fluorescence units to standardized units (MESF, ABC)

  • Establish clear positivity thresholds based on biological controls

• Quantitative analysis methods:

  • Move beyond binary positive/negative classifications to quantitative expression analysis

  • Implement density plots and contour plots for better visualization of expression patterns

  • Utilize histogram overlays to compare expression across conditions

  • Consider statistical approaches appropriate for flow cytometry data (often non-normally distributed)

• Multiparameter analysis strategies:

  • Implement dimensionality reduction techniques (tSNE, UMAP) for high-parameter datasets

  • Use clustering algorithms to identify populations with similar CD58 expression patterns

  • Consider machine learning approaches for identifying subtle expression differences

  • Integrate CD58 expression data with functional readouts for correlation analysis

• Visualization best practices:

  • Select color schemes that accommodate colorblind viewers

  • Maintain consistent axis scaling across comparable plots

  • Include appropriate statistical annotations

  • Provide sufficient technical details for reproducibility

• Integrated analysis frameworks:

  • Combine multiple data types (surface CD58, soluble CD58, functional outcomes)

  • Implement systems biology approaches for pathway analysis

  • Consider longitudinal analysis for temporal studies

  • Develop computational pipelines for consistent analysis across experiments