LILRB5 Antibody, FITC conjugated

What is LILRB5 and what are its primary functions in the immune system?

LILRB5 (Leukocyte Immunoglobulin-Like Receptor, Subfamily B, Member 5) is an inhibitory receptor belonging to the LILR family, also known as CD85c or LIR8. It contains transmembrane domains and immunoreceptor tyrosine-based inhibitory motifs (ITIMs).

Research indicates that LILRB5 functions primarily as a receptor for class I MHC antigens, with specific binding to HLA class I free heavy chains. Studies have shown that HLA-B27 free heavy chain (FHC) dimer binds to LILRB5-transfected cells, and this binding can be blocked with class I heavy chain antibody HC10 and anti-LILRB5 antisera . This unique binding specificity for HLA-class I heavy chains distinguishes LILRB5 from other LILR family members that typically bind to β2m-associated HLA-class I.

Additionally, LILRB5 has been implicated in mycobacterial recognition, with studies showing that its transcriptional profile is significantly up-regulated following exposure to mycobacteria. LILRB5 can trigger signaling through direct engagement with mycobacteria, suggesting a potential role during infection .

Which cell types express LILRB5 and how is expression regulated?

LILRB5 demonstrates a complex expression pattern across immune cell populations:

• Monocytes: Significant LILRB5 expression has been detected on the surface of CD14+ monocyte cells .

• T cells: More than 80% of CD4+ and CD8+ T cells express LILRB5, as well as a proportion of γδ T cells .

• Mast cells: Mature cord blood-derived mast cells (hMCs) express LILRB5 in cytoplasmic granules rather than on the cell surface .

• NK cells: Previous investigations identified LILRB5 mRNA in NK cells .

Flow cytometry analyses indicate that LILRB5 is not significantly expressed on the surface of B lymphocytes . Expression regulation appears to be tissue and context-dependent, with studies showing upregulation in response to mycobacterial challenge, particularly in cells from BCG-vaccinated individuals .

What experimental applications is LILRB5 Antibody, FITC conjugated commonly used for?

LILRB5 Antibody, FITC conjugated is suitable for multiple research applications:

• Flow Cytometry (FCM/FACS): The primary application, allowing detection of LILRB5 expression on immune cell populations. Typical working dilutions range from 1:20-100 .

• Immunofluorescence (IF):

  • IF in paraffin-embedded tissues (IHC-P): 1:50-200 dilution

  • IF in frozen sections (IHC-F): 1:50-200 dilution

  • IF in cell culture (ICC): 1:50-200 dilution

• Western Blotting (WB): Used at dilutions of 1:300-5000 depending on protein abundance .

• Intracellular staining: Particularly valuable for detecting LILRB5 in cellular compartments such as mast cell granules, using appropriate fixation and permeabilization protocols .

When selecting application parameters, researchers should consider that LILRB5 may be located either on the cell membrane or in intracellular compartments depending on the cell type being studied .

What are the optimal experimental conditions for detecting LILRB5 using FITC-conjugated antibodies in flow cytometry?

For optimal detection of LILRB5 using FITC-conjugated antibodies in flow cytometry, consider the following protocol elements:

Fixation and Permeabilization:

• For surface staining: 4% paraformaldehyde fixation is recommended

• For intracellular detection: 4% paraformaldehyde fixation followed by 90% methanol permeabilization has proven effective

Antibody Concentration and Controls:

• Optimal working concentration typically ranges from 1:20-100 dilution (approximately 0.5-2 μg/ml)

• Essential controls include:

  • Isotype control: Use a Rabbit monoclonal IgG (e.g., Rabbit IgG, monoclonal [EPR25A])

  • Unstained cells (without primary and secondary antibody incubation)

  • Secondary antibody-only control

Detection Strategy:

• Direct detection using FITC-conjugated anti-LILRB5

• For signal amplification in cells with low expression: Consider a two-step approach using unconjugated primary anti-LILRB5 followed by FITC-conjugated secondary antibody (e.g., Goat Anti-Rabbit IgG H&L Alexa Fluor® 488)

Gating Strategy:

• When analyzing peripheral blood subsets, use appropriate markers:

  • Monocytes: CD14+

  • T lymphocytes: CD3+, CD14-, CD19-

  • Further T cell subset identification: CD4+ (helper) vs. CD8+ (cytotoxic)

Researchers should note that expression patterns differ significantly between cell types, with monocytes showing predominantly surface expression while mast cells exhibit primarily intracellular localization .

How can I validate the specificity of LILRB5 antibody staining in my experiments?

Validating LILRB5 antibody specificity is crucial for reliable experimental results. A comprehensive validation strategy includes:

Expression System Controls:

• Transfected vs. non-transfected cells: Use 293T cells transfected with a human LILRB5 expression vector (containing a verification tag like myc) as a positive control alongside non-transfected 293T cells

• Co-staining with anti-myc tag antibody in transfected cells confirms expression of the transfected LILRB5 construct

Blocking Experiments:

• Pre-incubation with anti-LILRB5 antisera should specifically inhibit binding of LILRB5 ligands (e.g., B27 dimer tetramers) to LILRB5-expressing cells

• Control antibodies (normal goat serum) should not affect binding

Cross-Reactivity Testing:

• Confirm that the anti-LILRB5 antibody does not stain cells transfected with other LILR family members

• Flow cytometry screening with cells expressing LILRA1, LILRA4, LILRA5, LILRA6, and LILRB2 should not show positive staining with the LILRB5-specific antibody

Biochemical Validation:

• Co-immunoprecipitation of LILRB5 with its known ligands (HLA-class I heavy chains)

• Western blot detection of the immunoprecipitated complex using both anti-FLAG (for tagged LILRB5) and HC10 (for HLA-class I heavy chains) antibodies

Cell-Type Specific Expression Pattern:

• Confirm expected expression patterns across immune cell subsets

• LILRB5 should be detected primarily on monocytes, T cells (especially CD8+), and intracellularly in mast cells

What methodologies best capture LILRB5 expression changes during mycobacterial infection?

To effectively study LILRB5 expression dynamics during mycobacterial infection, researchers should employ a multi-technique approach:

Transcriptional Analysis:

• Real-time PCR is the primary method to detect transcriptional changes in LILRB5 following mycobacterial exposure

• Studies have shown significant up-regulation of LILRB5 transcripts after exposure to mycobacteria, particularly in cells from BCG-vaccinated individuals

Protein Expression Analysis:

• Flow cytometry with anti-LILRB5 antibodies to quantify changes in surface expression on different immune cell populations

• Intracellular staining protocols for detection in relevant cell compartments

• Western blotting of cell lysates to measure total protein levels

Functional Reporter Systems:

• Transfectant cells incorporating reporter systems (e.g., NFAT-GFP reporters) have successfully demonstrated LILRB5 signaling through direct engagement with mycobacteria

• This approach allows monitoring of receptor activation rather than just expression changes

Experimental Design Considerations:

• Compare cells from BCG-vaccinated versus unvaccinated individuals

• Test multiple mycobacterial species (M. tuberculosis, M. bovis, BCG)

• Include appropriate time course analyses (early vs. late responses)

• Examine different cell types (monocyte-derived DCs, macrophages, T cells)

Research has demonstrated that LILRB5 is the only LILR receptor significantly up-regulated in response to mycobacterial challenge, with this effect being more pronounced in cells derived from BCG-vaccinated donors .

How can I design experiments to investigate LILRB5's role in T cell proliferation?

To investigate LILRB5's role in T cell proliferation, researchers should consider the following experimental design strategies:

Cell Separation and Purification:

• Isolate primary T cell populations (CD4+ and CD8+) using magnetic bead separation or FACS sorting

• Verify purity by flow cytometry using appropriate markers

LILRB5 Ligation Approaches:

• Antibody-mediated crosslinking:

  • Pre-treat T cells with anti-LILRB5 antibody followed by crosslinking with anti-IgG secondary antibody

  • Include appropriate controls:

    • Secondary antibody alone (critical as secondary antibody alone can reduce CD8 T cell proliferation)

    • Isotype control antibody with secondary antibody

• Ligand-mediated stimulation:

  • Use purified HLA-class I free heavy chains (particularly B27 dimers) to engage LILRB5

  • Block with HC10 antibody to confirm specificity

Proliferation Assays:

• Allogeneic mixed lymphocyte reactions (MLRs) have successfully demonstrated LILRB5 effects

• Options for measuring proliferation:

  • CFSE dilution assay with flow cytometric analysis

  • Thymidine incorporation assays

  • Ki-67 staining

Analytical Considerations:

• Analyze CD4+ and CD8+ T cells separately, as research shows differential effects

• Compare LILRB5 ligation effects on T cells versus antigen-presenting cells

• Investigate potential mechanisms by measuring cytokine production, activation markers, and signaling pathway activation

Previous studies have shown that LILRB5 ligation significantly increases proliferation of CD8+ T cells compared to secondary antibody alone (p = 0.0049), while no such effect was observed in CD4+ T cells, demonstrating cell type-specific functions .

What are the current challenges in studying LILRB5 interactions with HLA class I heavy chains?

Researching LILRB5 interactions with HLA class I heavy chains presents several methodological challenges:

Unique Binding Properties:

• Unlike other LILR family members that primarily bind β2m-associated HLA-class I, LILRB5 specifically binds to HLA class I free heavy chains (FHCs)

• This binding specificity is likely due to differences in the D1 and D2 immunoglobulin-like binding domains of LILRB5

Technical Challenges in Ligand Preparation:

• Generating stable, properly folded HLA class I FHCs requires specialized techniques

• B27 FHC dimers are particularly challenging to prepare in their native conformation

• Researchers typically use tetramers of B27 FHC dimers for binding studies

Detection System Limitations:

• Demonstrating direct binding requires specialized approaches:

  • Flow cytometry with fluorescently labeled tetramers

  • Co-immunoprecipitation followed by western blotting

  • Surface plasmon resonance for binding kinetics

Verification of Binding Specificity:

• Critical controls include:

  • Pre-incubation with HC10 antibody (blocks class I heavy chains)

  • Pre-incubation with anti-LILRB5 antisera

  • Use of non-related HLA class I molecules

Structural Considerations:

• The binding interface between LILRB5 and HLA class I FHCs remains poorly characterized

• Determining which amino acid residues are critical for this interaction requires extensive mutagenesis studies

• The stoichiometry of binding (1:1 vs. multimeric complexes) is not fully understood

Research has demonstrated that the binding specificity of LILRB5 for HLA class I FHCs differs from other LILR family members, highlighting the need for specialized techniques when studying these interactions .

How does LILRB5 release from mast cells impact experimental design for studying inflammatory responses?

The discovery that LILRB5 is stored in cytoplasmic granules of mast cells and released upon FcεRI crosslinking introduces unique experimental considerations:

Detection of Soluble LILRB5:

• Supernatant analysis requires sensitive detection methods

• ELISA or multiplex bead-based assays can quantify released LILRB5

• Western blotting of concentrated supernatants provides visualization of the released protein

Kinetics and Stimulation Protocols:

• Time-course experiments are essential to establish release dynamics

• Optimal stimulation protocols for triggering LILRB5 release:

  • IgE sensitization followed by antigen cross-linking

  • Direct FcεRI crosslinking using anti-FcεRI antibodies

  • Comparison with non-IgE dependent stimuli (calcium ionophores, compound 48/80)

Functional Assessment of Released LILRB5:

• Co-culture systems can assess the effects of mast cell-derived soluble LILRB5 on other immune cells

• Recombinant soluble LILRB5 can serve as a control to validate biological activities

• Blocking experiments using anti-LILRB5 antibodies in the culture medium can confirm specificity

Potential Confounding Factors:

• Other granule contents released simultaneously may influence experimental outcomes

• Proteolytic processing of LILRB5 after release may alter its functional properties

• Local concentrations in tissues likely differ from in vitro conditions

Research suggests that released LILRB5 from mast cells may have potential for amplification of mast cell-dependent inflammatory responses, making it an important consideration when designing studies of allergic or inflammatory conditions .

What are the considerations when comparing LILRB5 expression data across different immune cell subsets?

When comparing LILRB5 expression across immune cell subsets, researchers should address several technical and biological variables:

Subcellular Localization Differences:

• T cells and monocytes: Predominantly surface expression (>80% of CD4+ and CD8+ T cells)

• Mast cells: Primarily intracellular in cytoplasmic granules

• NK cells: Expression originally reported at mRNA level only

Detection Method Variability:

• Flow cytometry protocols must be optimized for each cell type:

  • Surface staining (non-permeabilized) for monocytes and T cells

  • Intracellular staining (permeabilized) for mast cells

  • Combined surface/intracellular staining may be necessary for comprehensive analysis

Standardization Approaches:

• Quantitative comparisons require standardized controls:

  • Consider using antibody binding capacity (ABC) beads

  • Include consistent positive controls (e.g., transfected cell lines)

  • Report mean fluorescence intensity ratios rather than absolute values

Biological Factors Affecting Expression:

• Activation state can significantly alter LILRB5 expression

• Cell source and isolation methods may impact expression levels

• Mycobacterial exposure upregulates LILRB5, particularly in vaccinated individuals

Comprehensive Analysis Strategy:

• Multi-parameter flow cytometry with lineage markers

• Confirmation with transcript analysis (qPCR)

• Protein validation by western blotting

• Appropriate statistical analyses for comparing expression across populations

Research indicates substantial heterogeneity in LILRB5 expression and localization across different immune cell populations, necessitating careful methodological considerations when making comparisons .

How can researchers distinguish LILRB5 from other LILR family members in immunological assays?

Distinguishing LILRB5 from other LILR family members presents significant challenges due to structural similarities but is achievable through several approaches:

Antibody Selection and Validation:

• Use monoclonal antibodies specifically validated against multiple LILR family members

• Verify specificity by testing antibodies against cells transfected with different LILR proteins

• Anti-LILRB5 antibodies should not stain cells expressing LILRA1, LILRA4, LILRA5, LILRA6, or LILRB2

Epitope Mapping:

• Focus on antibodies targeting unique regions in LILRB5

• The most specific antibodies target regions outside the conserved Ig-like domains

• Commercially available antibodies targeting amino acids 151-250/590 or 24-350 of LILRB5 have demonstrated specificity

Functional Discrimination:

• LILRB5 uniquely binds HLA class I free heavy chains, unlike other LILRs that bind β2m-associated HLA class I

• Ligand binding assays can functionally discriminate LILRB5:

  • B27 FHC dimer tetramers bind LILRB5 but not other LILRs

  • This binding is specifically blocked by HC10 antibody and anti-LILRB5 antisera

Expression Pattern Analysis:

• Cell type-specific expression patterns help distinguish LILRs:

  • LILRB5 is highly expressed on T cells, unlike most other LILRs

  • LILRB5 is found in mast cell granules, a unique localization pattern

  • Monocytes express multiple LILRs, requiring careful discrimination

Molecular Approaches:

• RT-PCR with LILRB5-specific primers for transcript analysis

• siRNA knockdown to confirm antibody specificity

• CRISPR-Cas9 gene editing for definitive validation studies

Research demonstrates that careful antibody selection and validation, combined with functional binding assays, provide the most reliable methods for specifically identifying LILRB5 in complex immunological contexts .

What are the optimal storage and handling conditions for LILRB5 Antibody, FITC conjugated?

To maintain optimal activity of LILRB5 Antibody, FITC conjugated, researchers should follow these storage and handling guidelines:

Storage Temperature:

• Store at -20°C for long-term preservation

• Aliquot into multiple vials to avoid repeated freeze-thaw cycles

• Short-term storage (1-2 weeks) at 4°C is possible but not recommended for maintaining fluorescence intensity

Buffer Composition:

• Typical storage buffer contains:

  • 0.01M TBS (pH 7.4)

  • 1% BSA as a stabilizer

  • 0.03% Proclin300 as a preservative

  • 50% Glycerol to prevent freeze damage

Light Protection:

• FITC conjugates are light-sensitive

• Store in amber vials or wrapped in aluminum foil

• Minimize exposure to light during experimental procedures

• Work under reduced laboratory lighting when possible

Working Solution Preparation:

• Dilute antibody immediately before use

• Prepare working solutions in appropriate buffers (PBS with 0.5-1% BSA typically)

• For flow cytometry applications, filter buffer through 0.2μm filter to remove particulates

Quality Control Monitoring:

• Periodically check fluorescence intensity using positive control samples

• Be aware that multiple freeze-thaw cycles can reduce activity

• Typical shelf-life under recommended storage conditions is approximately 1 year

Proper storage and handling significantly impact experimental outcomes, particularly for fluorescence-based applications where signal intensity directly affects detection sensitivity .

How can I troubleshoot weak or non-specific staining with LILRB5 Antibody, FITC conjugated?

When encountering weak or non-specific staining with LILRB5 Antibody, FITC conjugated, a systematic troubleshooting approach is recommended:

Weak Signal Issues:

• Antibody Titration:

  • Test multiple antibody concentrations (1:20 through 1:200)

  • Optimal signal-to-noise ratio may require higher concentrations than manufacturer recommendations

• Cell Preparation Optimization:

  • For surface staining: Ensure viable cells with minimal dead cell contamination

  • For intracellular staining: Optimize fixation and permeabilization conditions

  • Test different permeabilization reagents (methanol vs. saponin-based)

• Signal Amplification Strategies:

  • Consider indirect staining using unconjugated primary antibody and FITC-conjugated secondary

  • Biotin-streptavidin systems may increase sensitivity

  • Tyramide signal amplification for immunohistochemistry applications

Non-specific Staining Issues:

• Blocking Optimization:

  • Increase blocking time (30-60 minutes)

  • Test different blocking reagents (5-10% normal serum, BSA, commercial blockers)

  • Include Fc receptor blocking reagents when analyzing Fc receptor-positive cells

• Background Reduction:

  • Include 0.1-0.5% detergent (Triton X-100, Tween-20) in washing buffers

  • Increase number and duration of washing steps

  • Pre-absorb antibody with cell/tissue lysates from negative control samples

• Specificity Controls:

  • Always include isotype control at same concentration as primary antibody

  • Use LILRB5-transfected versus non-transfected cells as controls

  • Consider antibody pre-absorption with recombinant LILRB5 protein

Instrument and Analysis Considerations:

• Optimize flow cytometer PMT voltages for FITC channel

• Check for and compensate spectral overlap with other fluorochromes

• Apply appropriate gating strategies to exclude dead cells and debris

Experimental evidence indicates that 4% paraformaldehyde fixation followed by 90% methanol permeabilization provides optimal conditions for detecting LILRB5 in flow cytometry and immunofluorescence applications .