TLR5 Antibody, FITC conjugated

What is the optimal concentration of FITC-conjugated TLR5 antibody for flow cytometry analysis?

The recommended concentration for FITC-conjugated TLR5 antibody (Clone: ABM22G1) in flow cytometry applications is 0.5-1 μg per 10^6 cells . This concentration range has been validated across multiple cell types including monocytes and macrophages. For optimal results, researchers should perform titration experiments on their specific cell populations, as expression levels may vary significantly between different tissue sources.

When analyzing TLR5 expression in synovial fluid samples or other inflammatory environments, consider the following optimization protocol:

For co-staining experiments, researchers should pair the TLR5-FITC antibody with appropriate markers such as FITC-conjugated CD8 for T cells or FITC-labeled anti-CD14 for monocyte/macrophage populations .

How should researchers prepare cells for optimal TLR5 detection?

Proper cell preparation is critical for accurate TLR5 detection. Based on published methodologies, the following protocol is recommended:

• Wash cells with FACS buffer (PBS containing 0.2% BSA and 0.1% sodium azide)

• Block non-specific binding using 50% human serum and 0.5% BSA for 15-20 minutes at room temperature

• Stain with anti-TLR5-FITC antibody at 0.5-1 μg/10^6 cells

• For co-staining applications, include lineage markers such as FITC-labeled anti-CD14 for monocytes

• Incubate at 4°C for 30 minutes in the dark

• Wash twice with FACS buffer before analysis

This methodology has been validated in studies examining TLR5 expression in rheumatoid arthritis and cancer research contexts .

What is the subcellular localization of TLR5 and how does this affect antibody binding?

TLR5 is primarily localized to the cell membrane . Its subcellular location is critical for its function as a pattern recognition receptor for flagellin. When detecting TLR5 using FITC-conjugated antibodies, researchers should consider:

• TLR5 is accessible on non-permeabilized cells, allowing surface staining

• TLR5 undergoes post-translational modification through phosphorylation at Tyr-798 upon flagellin binding

• This phosphorylation is required for downstream signaling

For comprehensive detection protocols that capture both surface and potentially internalized receptors following activation, researchers may need to include both surface and intracellular staining methods to track receptor trafficking following flagellin stimulation.

How does TLR5 expression vary across different tissues and disease states?

TLR5 exhibits distinct tissue-specific expression patterns that change during disease processes:

Immunohistochemical analysis of synovial tissues has shown that TLR5 is significantly upregulated in both rheumatoid arthritis (RA) and osteoarthritis (OA) compared to normal tissues. Specifically, TLR5 immunostaining was elevated in synovial tissue lining and sublining macrophages, and endothelial cells, with co-localization to CD68+ and VWF+ cells .

What are the key signaling pathways downstream of TLR5 activation and how can they be measured?

TLR5 signaling proceeds through several interconnected pathways that can be experimentally assessed:

• MyD88-dependent signaling: TLR5 signaling is dependent on MyD88 and IRAK1 adapter proteins

• Activation cascades: Subsequent activation occurs through:

  • NF-κB pathway

  • MAPK pathway

  • PI3K pathway

• Tyrosine phosphorylation: TLR5 is phosphorylated at Tyr-798 upon flagellin binding, which is required for signaling

Researchers can measure these pathways using:

A validated protocol for assessing TLR5 signaling involves treating cells with flagellin (50-100 ng/mL, <50EU/mg) and harvesting cells at 6 hours for mRNA analysis or 24 hours for protein detection by ELISA .

How can researchers assess TLR5-specific effects in complex cellular systems?

Given the overlapping nature of TLR signaling pathways, isolating TLR5-specific effects requires careful experimental design:

• Use TLR5-specific blocking antibodies (10 μg/mL) to inhibit flagellin binding

• Compare responses to flagellin with other TLR ligands to identify TLR5-specific signatures

• Employ siRNA knockdown of TLR5 to confirm specificity

• Consider using cells from TLR5-deficient models as negative controls

In studies examining synovial fluid-mediated TNF-α production, pre-treatment of monocytes with anti-TLR5 antibody (10 μg/mL) for 1 hour prior to stimulation reduced TNF-α transcription by approximately 80%, demonstrating the central role of TLR5 in this inflammatory response .

How does TLR5 contribute to inflammatory diseases like rheumatoid arthritis?

TLR5 plays a significant role in rheumatoid arthritis pathogenesis through several mechanisms:

• Elevated expression: TLR5 is significantly upregulated in RA synovial tissue lining and sublining macrophages and endothelial cells compared to normal tissues

• Disease correlation: TLR5 expression levels in RA monocytes strongly correlate with:

  • Disease Activity Score (DAS28)

  • TNF-α levels

• Inflammatory mediation: TLR5 is a critical modulator of TNF-α production in RA synovial fluid

• Reciprocal regulation: TNF-α exhibits feedback regulation with TLR5 expression in RA monocytes

• Cytokine modulation: In RA macrophages, TLR5 expression is induced by IL-17 and IL-8

These findings suggest that TLR5 may serve as a predictor for RA disease progression and a potential therapeutic target. Experimental models demonstrate that blocking TLR5 significantly reduces synovial fluid-mediated TNF-α transcription .

What methodological approaches can researchers use to study TLR5 in tumor microenvironments?

TLR5 has emerging roles in cancer biology, with several methodological approaches available for investigation:

• Engineered T cells: T cells can be engineered to secrete bacterial flagellin (TLR5 ligand) to reshape the tumor microenvironment

• Assessment parameters: When studying TLR5 in tumors, researchers should evaluate:

  • T cell infiltration

  • Chemokine receptor expression (CCR1, CXCR3)

  • Exhaustion markers (PD1, Lag3)

  • Myeloid-derived suppressor cell populations (CD11+Gr1+)

  • Chemokine/cytokine profiles

In xenograft models, tumor-reactive T cells engineered to deliver TLR5 ligand to the tumor site demonstrated potent antitumor responses compared to conventional T cells. Analysis revealed increased T cell infiltration, upregulated chemokine receptors, reduced inhibitory markers, and modifications to the myeloid compartment within the tumor microenvironment .

How can researchers design experiments to evaluate TLR5-mediated effects on immune cell functions?

When designing experiments to assess TLR5-mediated immune modulation, consider this systematic approach:

• Cell isolation and preparation:

  • For monocytes/macrophages: Isolate using magnetic bead selection

  • Block with 50% human serum and 0.5% BSA before staining

  • Validate populations with CD14 co-staining

• Stimulation protocols:

  • Use flagellin at 50-100 ng/mL (ensure <50EU/mg endotoxin)

  • For blocking studies: Pretreat with anti-TLR5 antibody (10 μg/mL) for 1 hour

  • Include appropriate controls (IgG isotype)

• Readout systems:

  • Assess proliferation using standard methods (MTT, thymidine incorporation)

  • Measure cytokine production at both mRNA (6h) and protein levels (24h)

  • Key cytokines: TNF-α, IL-6, CCL2

• Validating specificity:

  • Compare TLR5 responses to other TLR ligands (TLR2, TLR4)

  • Evaluate responses in cells with known differential TLR5 expression

This approach has successfully demonstrated that TLR5 stimulation increases proliferation of human CD8+ T cells comparable to CD28 costimulation, enhances cytokine production, and augments cytolytic activity against target cells .

What are the methodological considerations for using TLR5 ligand-secreting T cells in immunotherapy research?

Engineering T cells to secrete TLR5 ligands represents an innovative approach to immunotherapy with specific methodological requirements:

• T cell engineering protocol:

  • Isolate T cells and stimulate with appropriate stimuli (e.g., HLA-A2/MART-1:27-35 and anti-CD28 for melanoma-reactive T cells)

  • Maintain cells with 100 U/mL IL-2

  • Transduce with vectors encoding TLR5 ligand (flagellin)

  • Confirm secretion by Western blot using anti-flagellin antibodies

• Validation of TLR5L secretion:

  • Harvest culture supernatants

  • Concentrate using centrifugal concentrators

  • Perform SDS-PAGE and Western blot with anti-flagellin antibody

  • Detect using enhanced chemiluminescence

• In vivo experimental design:

  • Establish tumor models (e.g., 1×10^5 B16-F1 cells subcutaneously)

  • Allow tumors to reach approximately 50mm^2

  • Consider pre-conditioning with radiation (400 cGy)

  • Administer engineered T cells intravenously (2.5×10^6 cells)

  • Measure tumor growth kinetics and survival

This approach has demonstrated significant antitumor effects in both xenogeneic and syngeneic models, with increased T cell infiltration and favorable modifications to the tumor microenvironment .

How can researchers troubleshoot common issues with FITC-conjugated TLR5 antibody staining?

When working with FITC-conjugated TLR5 antibodies, researchers may encounter several technical challenges:

For optimal results when assessing TLR5 expression across different tissues, use standardized protocols for tissue fixation and processing. In immunohistochemistry applications, a validated protocol involves incubating with anti-TLR5 antibody (1:50 dilution) and scoring lining, sublining macrophages, and endothelial cell staining on a 0-5 scale .

What reference standards and controls are essential for TLR5 research?

Proper controls are critical for reliable TLR5 research:

• Positive controls:

  • Cell lines with confirmed high TLR5 expression (e.g., monocytes, intestinal epithelial cells)

  • Tissues with known TLR5 expression (ovary, peripheral blood leukocytes)

• Negative controls:

  • Isotype-matched control antibodies (Mouse IgG2a Kappa for ABM22G1 clone)

  • Cells with low/absent TLR5 expression

• Validation controls:

  • Parallel staining with multiple TLR5 antibody clones

  • Co-localization with lineage markers (CD68 for macrophages, VWF for endothelial cells)

  • TLR5 knockdown or knockout samples where available

For comprehensive analysis of TLR5+ cells in complex samples, researchers should use multiparameter flow cytometry combining TLR5-FITC with lineage markers such as CD14 for monocytes/macrophages .