TMOD1 Antibody, FITC conjugated

What is TMOD1 and what cellular functions does it regulate?

TMOD1 (Tropomodulin1) is an actin-binding protein that caps the slow-growing end of actin filaments (F-actin), decreasing the rate of actin depolymerization. It also binds to G-actin and promotes actin nucleation . TMOD1 was first isolated from human erythrocytes and is expressed in various cell types including cardiomyocytes, lens fiber cells, neurons, and immune cells such as monocytes and dendritic cells .

In immune cells, TMOD1 plays critical roles in regulating maturation and immune functions. Research demonstrates that TMOD1 is indispensable for controlling dendritic cell maturation by regulating TLR4 signaling and actin cytoskeleton organization . Additionally, in macrophages, TMOD1 modulates inflammatory responses by regulating TLR4 endocytosis and intracellular trafficking .

How does TMOD1 expression change during immune cell maturation?

TMOD1 expression significantly increases upon maturation in dendritic cells. Quantitative PCR data has shown that TMOD1 is upregulated approximately 4-fold in LPS-stimulated mature dendritic cells (mDCs) compared to immature dendritic cells (imDCs) . This upregulation was further confirmed by Western blotting, which demonstrated that while TMOD1 is present at lower levels in imDCs, its expression increases substantially upon DC maturation .

The upregulation of TMOD1 during maturation appears to be a result of the activation of the NF-κB signaling pathway. Importantly, this differential expression pattern correlates with distinct functions in immature versus mature dendritic cells, suggesting that TMOD1 plays specialized roles depending on the activation state of the cell .

What applications is a FITC-conjugated TMOD1 antibody suitable for?

A FITC-conjugated TMOD1 antibody is particularly valuable for applications requiring fluorescence detection. Based on experimental protocols described in research studies, suitable applications include:

• Flow cytometry: For quantitative analysis of TMOD1 expression in different cell populations and subsets. Flow cytometric analysis has been used successfully to measure surface marker expression in TMOD1-expressing cells .

• Immunofluorescence microscopy: For visualizing TMOD1 localization within cells and tissues, particularly in relation to actin cytoskeleton components.

• Multi-parameter analysis: FITC conjugation allows for incorporation into multi-color panels alongside other markers to characterize cell phenotypes.

• Live-cell imaging: For monitoring dynamic changes in TMOD1 distribution during cellular processes.

The FITC fluorophore's excitation/emission characteristics (approximately 495nm/519nm) make it compatible with standard fluorescence detection equipment, providing good sensitivity for endogenous TMOD1 detection .

What is the molecular weight of TMOD1 and how should researchers validate antibody specificity?

TMOD1 has a molecular weight of approximately 45 kDa as detected by Western blotting . When validating a FITC-conjugated TMOD1 antibody, researchers should:

• Perform Western blotting using positive control lysates from cells known to express TMOD1 (e.g., mature dendritic cells) to confirm binding to a protein of the expected molecular weight.

• Include negative controls such as TMOD1-deficient cells (e.g., TMOD1 knockout or knockdown models).

• Conduct parallel staining with established, validated TMOD1 antibodies from different sources or clones.

• Implement appropriate isotype controls in flow cytometry and immunofluorescence applications to assess non-specific binding.

The antibody should demonstrate consistent specificity across different experimental conditions and cell types that express varying levels of TMOD1 .

How does TMOD1 deficiency affect dendritic cell function and what methodologies can detect these changes?

TMOD1 deficiency significantly impacts multiple aspects of dendritic cell function. Research utilizing TMOD1-deficient dendritic cells has revealed:

• Reduced surface expression of costimulatory molecules (CD86, MHC-II, CD80, CD40) and chemokine receptors (CCR7), indicating impaired maturation .

• Decreased secretion of inflammatory cytokines including TNF-α, IL-12(p70), and IFN-γ, but increased production of IFN-β and IL-10 .

• Impaired random and chemotactic migration capabilities critical for DC function .

• Deteriorated T-cell stimulatory ability in mixed lymphocyte reaction assays .

• Reduced F-actin content and decreased cell stiffness, affecting cellular biomechanics .

These changes can be methodologically detected through:

• Flow cytometry with fluorescently-labeled antibodies against surface markers (CD11c, CD86, MHC-II, CD80, CD40, CCR7)

• ELISA or cytometric bead arrays for quantifying secreted cytokines

• Modified Boyden chamber assays for migration assessment

• MLR (mixed lymphocyte reaction) assays to evaluate T-cell stimulation capacity

• F-actin quantification using phalloidin staining

• Atomic force microscopy for cell stiffness measurements

A FITC-conjugated TMOD1 antibody would be particularly valuable for multi-parameter flow cytometry and immunofluorescence experiments studying these phenotypic changes.

What is the relationship between TMOD1 and TLR4 signaling pathways?

TMOD1 plays a critical regulatory role in TLR4 signaling by differentially affecting the MyD88-dependent and TRIF-dependent pathways. The relationship is complex and multifaceted:

In dendritic cells, TMOD1 deficiency leads to:

• Decreased activity of MyD88-dependent NFκB and MAPK pathways

• Increased activity of the TRIF/IRF3 pathway

• Altered balance between inflammatory (TNF-α, IL-12) and regulatory (IFN-β, IL-10) cytokine production

In macrophages, TMOD1:

• Inhibits LPS-induced TLR4 endocytosis and intracellular trafficking

• Promotes increased surface TLR4 expression

• Enhances MyD88-dependent pathway activation

• Downregulates TRIF-dependent pathway signaling

• Modulates inflammatory response through the CD14/Syk/PLCγ2/IP3/Ca²⁺ signaling pathway

Mechanistically, TMOD1 influences TLR4 signaling by regulating actin cytoskeleton reorganization and membrane tension, which affects receptor trafficking and compartmentalization . Rescue experiments with exogenous TMOD1 have demonstrated reversibility of these effects, confirming the direct regulatory role of TMOD1 in TLR4 signaling .

How can researchers design experiments to investigate TMOD1's role in actin cytoskeleton dynamics during immune responses?

To investigate TMOD1's role in actin cytoskeleton dynamics during immune responses, researchers can implement a comprehensive experimental design strategy:

• Visualization techniques:

  • Use FITC-conjugated TMOD1 antibody alongside fluorescently-labeled phalloidin (for F-actin) to track co-localization during immune cell activation

  • Employ live-cell imaging to monitor dynamic changes in TMOD1 distribution relative to actin rearrangements during cell activation, migration, and immunological synapse formation

• Functional manipulation approaches:

  • Generate TMOD1 knockout or knockdown cells using CRISPR/Cas9 or siRNA technology

  • Conduct rescue experiments with wild-type or mutant TMOD1 to identify critical functional domains

  • Perform site-directed mutagenesis of TMOD1's actin-binding domains to assess domain-specific functions

• Quantitative assessments:

  • Measure F-actin content in TMOD1-sufficient and TMOD1-deficient cells following immune stimulation

  • Quantify actin polymerization/depolymerization rates in response to immune activation

  • Assess membrane tension and cellular stiffness using biophysical techniques like atomic force microscopy

• Signaling pathway analysis:

  • Evaluate TLR4 pathway activation in relation to actin cytoskeleton changes

  • Investigate the impact of pharmacological disruption of actin on TMOD1-mediated effects

These experimental approaches should be complemented with appropriate statistical analyses and controls to ensure robust and reproducible results.

What protocols are optimal for using FITC-conjugated TMOD1 antibody in studying immune cell subpopulations?

For optimal use of FITC-conjugated TMOD1 antibody in studying immune cell subpopulations, researchers should follow these methodological guidelines:

Sample Preparation Protocol:

• Isolate cells of interest (e.g., bone marrow-derived dendritic cells, peripheral blood mononuclear cells)

• For surface marker staining, wash cells in PBS with 1% BSA

• For intracellular TMOD1 staining, fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer for 10 minutes

Antibody Staining Protocol:

• Block non-specific binding with 5% normal serum from the same species as secondary antibodies

• Incubate with FITC-conjugated TMOD1 antibody at optimized dilution (typically 1:100 to 1:1000)

• For multi-parameter analysis, include additional fluorescently-labeled antibodies against relevant markers (CD11c, CD86, MHC-II, CD80, CD40, CCR7)

• Include appropriate isotype controls and single-stained samples for compensation

Flow Cytometry Analysis Approach:

• Implement logical gating strategy starting with FSC/SSC to identify cells of interest

• Use viability dye to exclude dead cells

• Apply FMO (Fluorescence Minus One) controls to accurately set gates for TMOD1-positive populations

• Correlate TMOD1 expression with other cellular markers to identify distinct subpopulations

• Quantify both percentage of positive cells and mean fluorescence intensity

These protocols should be optimized for each specific experimental setup and cell type being investigated.

How does TMOD1 contribute to inflammation regulation and what are the therapeutic implications?

TMOD1 significantly influences inflammatory regulation through multiple mechanisms, with important therapeutic implications:

In dendritic cells:

• TMOD1-deficient dendritic cells show reduced expression of costimulatory molecules and inflammatory cytokines

• TMOD1-deficient cells secrete higher levels of anti-inflammatory cytokines (IFN-β and IL-10)

• In an experimental autoimmune encephalomyelitis (EAE) mouse model, TMOD1-deficient dendritic cells induced immune tolerance and reduced disease severity

In macrophages:

• TMOD1 exacerbates inflammatory responses by enhancing the MyD88-dependent pathway

• TMOD1 deficiency relieves inflammatory response in LPS-induced acute lung injury mouse model

• TMOD1 influences inflammatory responses by modulating TLR4 endocytosis and signaling pathway selection

These findings suggest that TMOD1 may be a potential therapeutic target for:

• Treating excessive inflammation in conditions like sepsis and acute lung injury

• Modulating immune responses in autoimmune diseases

• Enhancing or suppressing specific aspects of dendritic cell function for immunotherapy applications

By targeting TMOD1, it may be possible to shift the balance between pro-inflammatory and anti-inflammatory pathways to achieve desired therapeutic outcomes. The development of small molecule inhibitors or other approaches to modulate TMOD1 function represents a promising avenue for future immunotherapeutic strategies .

What are the optimal controls when using FITC-conjugated TMOD1 antibody in flow cytometry?

When using FITC-conjugated TMOD1 antibody in flow cytometry experiments, implementing proper controls is essential for generating reliable and interpretable data:

Essential Controls:

• Isotype Control: Include an isotype-matched FITC-conjugated antibody of irrelevant specificity to assess non-specific binding and establish background fluorescence levels.

• Unstained Control: Cells processed without any antibody to determine autofluorescence levels of the cell population.

• FMO (Fluorescence Minus One) Control: In multicolor panels, include samples with all fluorochromes except FITC to accurately set TMOD1-positive gates.

• Biological Controls:

  • Positive control: Cells known to express high levels of TMOD1 (e.g., mature dendritic cells)

  • Negative control: Cells with minimal TMOD1 expression or TMOD1-deficient cells

• Compensation Controls: Single-stained samples for each fluorochrome to correct for spectral overlap in multicolor panels.

These controls should be processed identically to experimental samples, including all fixation and permeabilization steps, to ensure proper comparison .

How can researchers quantitatively assess TMOD1's impact on actin cytoskeleton organization?

To quantitatively assess TMOD1's impact on actin cytoskeleton organization, researchers can employ a combination of imaging and biochemical approaches:

Imaging-Based Quantification Methods:

• Confocal Microscopy with Digital Image Analysis:

  • Stain cells with phalloidin to visualize F-actin alongside FITC-conjugated TMOD1 antibody

  • Measure parameters such as F-actin content, stress fiber formation, and cortical actin distribution

  • Quantify colocalization between TMOD1 and F-actin using Pearson's or Mander's correlation coefficients

• Super-Resolution Microscopy:

  • Employ techniques like STORM or PALM to visualize nanoscale organization of actin filaments

  • Measure filament length, branching patterns, and spatial relationship with TMOD1

Biochemical and Biophysical Methods:

• F-actin/G-actin Ratio Determination:

  • Use differential centrifugation to separate F-actin and G-actin fractions

  • Quantify by Western blotting in TMOD1-sufficient versus TMOD1-deficient cells

• Cellular Mechanics Measurements:

  • Atomic force microscopy to assess cell stiffness and membrane tension

  • Micropipette aspiration to measure whole-cell mechanical properties

• Actin Dynamics Assays:

  • Fluorescence recovery after photobleaching (FRAP) to measure actin turnover rates

  • Live cell imaging with fluorescently labeled actin to track polymerization dynamics

Research has shown that TMOD1-deficient dendritic cells exhibit reduced F-actin content and decreased cell stiffness, demonstrating TMOD1's crucial role in maintaining proper actin cytoskeleton organization and cellular biomechanics .

What troubleshooting approaches can resolve common issues with FITC-conjugated antibody detection?

When working with FITC-conjugated TMOD1 antibodies, researchers may encounter several technical challenges. Here are effective troubleshooting approaches for common issues:

Problem: Weak or Absent Signal

• Solution 1: Optimize antibody concentration through titration experiments

• Solution 2: Ensure proper fixation and permeabilization; TMOD1 may require specific permeabilization conditions to access intracellular binding sites

• Solution 3: Verify sample preparation conditions; excessive exposure to light can photobleach FITC

• Solution 4: Check for proper instrument settings; ensure correct excitation/emission filter configuration

Problem: High Background or Non-specific Staining

• Solution 1: Increase blocking time/concentration (5-10% serum for 30-60 minutes)

• Solution 2: Optimize antibody dilution; excessive antibody can increase non-specific binding

• Solution 3: Add 0.1% Tween-20 to wash buffers to reduce non-specific interactions

• Solution 4: Perform additional washing steps to remove unbound antibody

Problem: Autofluorescence Interference

• Solution 1: Include unstained controls to determine autofluorescence levels

• Solution 2: Use spectral unmixing algorithms in flow cytometry or confocal microscopy

• Solution 3: Consider alternative fluorophores if tissue/cell autofluorescence overlaps significantly with FITC spectrum

Problem: Inconsistent Results Between Experiments

• Solution 1: Standardize all protocols including fixation time, permeabilization conditions, and antibody incubation

• Solution 2: Prepare fresh fixatives and buffers for each experiment

• Solution 3: Include internal controls in each experiment for normalization

• Solution 4: Minimize freeze-thaw cycles of the antibody

These troubleshooting approaches can help resolve technical issues and ensure reliable detection of TMOD1 using FITC-conjugated antibodies .