LECT2 Antibody, FITC conjugated

What is LECT2 and why is it significant in immunological research?

LECT2 (Leukocyte cell-derived chemotaxin-2) is a multifunctional cytokine that plays critical roles in immune regulation. It possesses neutrophil chemotactic activity and functions as a positive regulator of chondrocyte proliferation . More importantly, LECT2 has been identified as a protective factor in bacterial sepsis by enhancing macrophage functions through the CD209a receptor . LECT2 does not exhibit metalloendopeptidase activity despite earlier predictions based on its structure . The significance of LECT2 in immunological research stems from its capacity to improve protective immunity in bacterial sepsis models, potentially offering novel therapeutic approaches for sepsis and other immune-related conditions .

What are the primary applications for FITC-conjugated LECT2 antibodies?

FITC-conjugated LECT2 antibodies serve multiple research applications:

• Flow cytometry: For quantifying LECT2-expressing cells in complex populations

• Immunofluorescence microscopy: For visualizing LECT2 distribution in tissues and cells

• Tracking LECT2-CD209a interactions: For studying receptor-ligand binding dynamics

• Phagocytosis assays: For monitoring macrophage activation following LECT2 stimulation

The FITC conjugation provides a bright fluorescent signal (excitation ~495nm, emission ~519nm) that enables direct visualization without secondary detection reagents. This is particularly valuable when studying LECT2's role in macrophage activation and phagocytosis, as demonstrated in research showing LECT2 enhances macrophage phagocytic ability and bacterial killing .

How does FITC conjugation affect LECT2 antibody performance?

FITC conjugation can influence antibody performance in several ways:

• Epitope accessibility: The conjugation process may affect antibody binding if FITC molecules attach near the antigen-binding site.

• Signal stability: FITC is susceptible to photobleaching, requiring careful handling during fluorescence microscopy or flow cytometry.

• pH sensitivity: FITC fluorescence decreases significantly below pH 7.0, which may impact experiments in acidic environments.

• Protein modification: The isothiocyanate reactive group of FITC reacts with primary amines at lysine residues and at the antibody's amino terminus, potentially altering protein structure .

For optimal results, researchers should validate FITC-conjugated LECT2 antibodies against unconjugated versions to ensure conjugation hasn't compromised antigen recognition capability.

What controls should be included when using FITC-conjugated LECT2 antibodies?

Essential controls for FITC-conjugated LECT2 antibody experiments include:

• Isotype control: FITC-conjugated antibody of the same isotype but irrelevant specificity to assess non-specific binding

• Blocking control: Pre-incubation with unconjugated LECT2 antibody or recombinant LECT2 protein

• Unstained control: To establish autofluorescence baseline

• LECT2 knockout/knockdown samples: To validate antibody specificity

• Positive control: Known LECT2-expressing tissues/cells (e.g., liver samples)

When studying LECT2's role in macrophage activation, include CD209a-deficient macrophages as additional controls, as research has shown CD209a mediates LECT2's effects on macrophage function .

What sample preparation techniques optimize FITC-conjugated LECT2 antibody performance?

Optimal sample preparation for FITC-conjugated LECT2 antibody applications includes:

• Fresh sample collection: Minimize protein degradation that could affect epitope recognition

• Gentle fixation: Use 2-4% paraformaldehyde to preserve cellular architecture while maintaining epitope accessibility

• Permeabilization: For intracellular LECT2 detection, use mild detergents (0.1-0.5% Triton X-100 or 0.1% saponin)

• Blocking: Use 5-10% serum from the species unrelated to the antibody source to reduce non-specific binding

• Protection from light: Minimize FITC photobleaching by covering samples and working in subdued lighting

• Anti-fading mounting media: For immunofluorescence microscopy to preserve signal intensity

These techniques help maintain both sample integrity and FITC conjugate stability, ensuring optimal signal-to-noise ratios in experimental results.

How can FITC-conjugated LECT2 antibodies help investigate LECT2-CD209a interactions?

FITC-conjugated LECT2 antibodies provide powerful tools for investigating LECT2-CD209a interactions through several sophisticated approaches:

• Co-localization studies: FITC-conjugated LECT2 antibodies can be used alongside CD209a-specific antibodies labeled with spectrally distinct fluorophores to visualize co-localization in cells and tissues. Research has demonstrated that LECT2 specifically interacts with the carbohydrate recognition domain (CRD) of CD209a, but not CD209b .

• FRET analysis: When paired with appropriate acceptor fluorophore-conjugated CD209a antibodies, FITC-conjugated LECT2 antibodies enable Förster Resonance Energy Transfer studies to quantify molecular proximity between LECT2 and CD209a.

• Immunoprecipitation validation: Using FITC-conjugated LECT2 antibodies to detect co-immunoprecipitated complexes can confirm protein-protein interactions identified through other methods. Previous research confirmed LECT2-CD209a interactions through co-immunoprecipitation in HEK293T cells expressing both proteins .

• Competitive binding assays: Researchers can use FITC-conjugated LECT2 antibodies to develop competitive binding assays that assess whether other molecules compete with CD209a for LECT2 binding.

These approaches can further elucidate the molecular basis for LECT2's specific interaction with CD209a, which has been shown to be essential for LECT2-mediated enhancement of macrophage function in bacterial sepsis models .

What methodological considerations are important for quantifying LECT2 expression using FITC-conjugated antibodies?

Accurate quantification of LECT2 expression using FITC-conjugated antibodies requires several methodological considerations:

For accurate LECT2 quantification, researchers should:

• Establish a standard curve using recombinant LECT2 protein

• Apply appropriate statistical methods for comparing expression levels between experimental groups

• Consider the cellular localization context when interpreting results, as LECT2 is secreted and may localize to different cellular compartments

• Account for FITC photobleaching in time-course experiments

Notably, existing research has employed Western blot to detect LECT2 in HEK-293T cell lysates transfected with LECT2 cDNA, demonstrating a predicted band size of 16 kDa .

How can researchers optimize FITC-conjugated LECT2 antibodies for dual/multi-color flow cytometry?

Optimizing FITC-conjugated LECT2 antibodies for dual/multi-color flow cytometry requires careful attention to several technical parameters:

• Panel design:

  • Select fluorophores with minimal spectral overlap with FITC (excitation ~495nm, emission ~519nm)

  • Consider brightness hierarchy, placing FITC on abundant targets or those requiring high sensitivity

  • Use fluorophore brightness compensation when studying less abundant targets

• Compensation strategy:

  • Prepare single-color controls with the exact antibody conjugates used in the full panel

  • Include an unstained control for autofluorescence determination

  • Apply compensation matrices before analysis to correct for spectral overlap

• Titration optimization:

  • Perform antibody titration experiments to determine optimal concentration (highest signal-to-noise ratio)

  • Use consistent staining conditions (temperature, duration, buffer composition)

  • Validate specificity using LECT2-deficient samples or blocking experiments

• Data analysis refinement:

  • Implement consistent gating strategies based on fluorescence-minus-one (FMO) controls

  • Consider alternative LECT2 antibody conjugates if FITC causes problematic spectral overlap

  • Apply appropriate statistical methods for comparing LECT2 expression between populations

When studying macrophage activation by LECT2, combining FITC-conjugated LECT2 antibodies with markers for phagocytosis (e.g., fluorescent E. coli or FluoSpheres) can provide valuable insights into functional responses, as research has shown LECT2 treatment enhances macrophage phagocytosis of E. coli by approximately 2-fold .

What experimental approaches can validate the specificity of FITC-conjugated LECT2 antibodies?

Validating the specificity of FITC-conjugated LECT2 antibodies requires multiple complementary approaches:

• Genetic validation:

  • Compare staining between wild-type and LECT2 knockout models

  • Use siRNA/shRNA knockdown of LECT2 in relevant cell lines

  • Test antibody in CD209a-deficient models, as CD209a mediates LECT2 functions

• Biochemical validation:

  • Pre-absorb antibody with recombinant LECT2 protein before staining

  • Perform Western blot analysis to confirm detection of appropriately sized protein (16 kDa)

  • Compare multiple antibody clones targeting different LECT2 epitopes

• Functional validation:

  • Correlate antibody staining with functional readouts (e.g., phagocytosis enhancement)

  • Neutralize LECT2 activity with the antibody and assess functional consequences

  • Compare antibody detection with alternative methods (e.g., mass spectrometry)

• Cross-reactivity assessment:

  • Test the antibody against related proteins to ensure specificity

  • Evaluate performance across multiple tissue types with known LECT2 expression patterns

  • Compare results across multiple detection platforms (flow cytometry, microscopy, Western blot)

Research has demonstrated that LECT2 specifically interacts with CD209a but not CD209b, highlighting the importance of validation in systems with appropriate positive and negative controls .

How can FITC-conjugated LECT2 antibodies be used to study LECT2's role in sepsis and infection models?

FITC-conjugated LECT2 antibodies offer powerful approaches for investigating LECT2's role in sepsis and infection models:

• Cellular distribution analysis:

  • Track LECT2-expressing cells during infection progression

  • Monitor changes in LECT2 receptor (CD209a) expression on macrophages

  • Quantify LECT2 production in various tissues during sepsis development

• Functional response assessment:

  • Correlate LECT2 expression with macrophage activation markers

  • Use FITC-conjugated LECT2 antibodies alongside phagocytosis assays to link expression with function

  • Track LECT2 binding to macrophages in relation to bactericidal activity

• Therapeutic intervention monitoring:

  • Assess how exogenous LECT2 treatment affects endogenous LECT2 production

  • Monitor changes in LECT2 expression following antimicrobial therapy

  • Track LECT2 expression in different macrophage populations during recovery from sepsis

• Mechanistic investigations:

  • Use FITC-conjugated LECT2 antibodies with CD209a visualization to study receptor-ligand interactions

  • Combine with complement component C3 staining to investigate LECT2's role in complement activation

  • Pair with cytokine profiling to correlate LECT2 expression with G-CSF, IFN-γ, and other beneficial mediators

Research has demonstrated that LECT2 treatment effectively improved the outcome in mouse models of sepsis by enhancing macrophage phagocytosis and bacterial killing via CD209a receptor interaction . FITC-conjugated LECT2 antibodies would enable researchers to track these processes with cellular and subcellular resolution.

What technical challenges exist when using FITC-conjugated LECT2 antibodies, and how can they be overcome?

FITC-conjugated LECT2 antibodies present several technical challenges that can be addressed through specific methodological approaches:

To optimize signal detection of LECT2 in macrophages, researchers should consider:

• Using CD209a expression as a guide for identifying LECT2-responsive cells, as research has shown CD209a mediates LECT2's effects on macrophage function

• Incorporating C3 blocking experiments to distinguish direct LECT2 effects from those mediated by complement activation, as LECT2 has been shown to enhance C3 production

• Developing novel quantitative assays that combine FITC-conjugated LECT2 antibody staining with functional readouts of macrophage activation