IL18R1 Antibody, FITC conjugated

What is IL18R1 and why is it an important research target?

IL18R1 (Interleukin 18 Receptor 1) is a cytokine receptor belonging to the interleukin 1 receptor family that specifically binds interleukin 18 (IL18) and is essential for IL18-mediated signal transduction. This receptor plays a critical role in the immune system, particularly in inflammatory and immune responses. IL18R1 is expressed on several immune cell types, including Th1 cells, NK cell subsets, neutrophils, and IL-12-stimulated tonsillar B cells. The receptor's significance lies in its role in the innate and adaptive immune responses, making it an important target for immunological research, particularly in studying inflammatory diseases, cancer, and autoimmune conditions . IL18R1's interaction with IL18 is part of a complex signaling pathway that influences cytokine production and immune cell activation, which has implications for various pathological conditions.

What are the key structural and functional characteristics of FITC-conjugated IL18R1 antibodies?

FITC-conjugated IL18R1 antibodies consist of immunoglobulins that specifically recognize and bind to IL18R1, with fluorescein isothiocyanate (FITC) molecules chemically attached to enable fluorescent detection. These antibodies typically target specific amino acid sequences within the IL18R1 protein. For instance, some antibodies target amino acids 21-120 of the receptor . When the antibody binds to IL18R1, the FITC component emits green fluorescence when excited at 488 nm, with emission at approximately 520 nm . The antibody's binding specificity is determined by the epitope it recognizes, while its functionality depends on factors including the host species, clonality (monoclonal vs. polyclonal), and the particular region of IL18R1 it targets. These characteristics influence the antibody's performance in various applications such as flow cytometry, immunofluorescence, and Western blotting. The fluorescent conjugation enables direct visualization of IL18R1 expression without requiring secondary detection reagents.

What are the optimal protocols for using FITC-conjugated IL18R1 antibodies in flow cytometry?

For optimal flow cytometry results with FITC-conjugated IL18R1 antibodies, researchers should follow a methodical approach. Begin with freshly isolated cells or properly maintained cell lines in a single-cell suspension at a concentration of 10^5 to 10^8 cells per test. Pre-titrated antibodies like the monoclonal H44 clone can be used at 5 μL (0.25 μg) per test in a final volume of 100 μL . The staining procedure should involve: (1) Washing cells in cold flow cytometry buffer (PBS with 1-2% FBS and 0.1% sodium azide); (2) Blocking Fc receptors for 10-15 minutes to reduce non-specific binding, particularly important when working with primary immune cells; (3) Incubating with the FITC-conjugated IL18R1 antibody at 4°C for 30 minutes in the dark; (4) Washing cells 2-3 times with buffer to remove unbound antibody; and (5) Resuspending in buffer containing a viability dye for analysis. When using a blue laser (488 nm), optimize detection settings for FITC emission at 520 nm . For multiparameter analysis, proper compensation is crucial to account for spectral overlap between FITC and other fluorophores. Include appropriate isotype controls to establish baseline fluorescence and single-stained controls for compensation. For optimal detection of IL18R1, consider that expression can be modulated by cytokines, particularly IFN-alpha and IL12, which induce expression in NK and T cells .

How can researchers optimize immunofluorescence techniques using FITC-conjugated IL18R1 antibodies for both cultured cells and tissue sections?

Optimizing immunofluorescence with FITC-conjugated IL18R1 antibodies requires distinct approaches for cultured cells and tissue sections. For cultured cells (IF (cc)), begin with cells grown on coverslips at 60-70% confluence. Fix with 4% paraformaldehyde for 15 minutes at room temperature, followed by membrane permeabilization with 0.1-0.2% Triton X-100 for intracellular epitopes. Block with 5% normal serum from the same species as the secondary antibody for 1 hour. Apply the FITC-conjugated IL18R1 antibody at an optimized dilution (typically starting at 1:50-1:200) and incubate overnight at 4°C in a humidified chamber . For paraffin-embedded tissue sections (IF (p)), proper deparaffinization and antigen retrieval are critical. After deparaffinization with xylene and rehydration through an ethanol series, perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). The choice of retrieval buffer should be empirically determined for IL18R1 detection . Following antigen retrieval, block endogenous peroxidase activity with 3% hydrogen peroxide and proceed with blocking and antibody incubation as with cultured cells. For both applications, counterstain nuclei with DAPI, mount with anti-fade mounting medium, and store slides at 4°C protected from light. To optimize signal-to-noise ratio, titrate the antibody concentration, adjust incubation times, and incorporate thorough washing steps. Include appropriate controls: negative controls (omitting primary antibody) and positive controls (tissues or cells known to express IL18R1, such as Th1 cells or IL-12-stimulated B cells) .

How can researchers accurately quantify IL18R1 expression levels using FITC-conjugated antibodies in flow cytometry?

Accurate quantification of IL18R1 expression using FITC-conjugated antibodies in flow cytometry requires a systematic approach. Researchers should first establish optimal antibody concentration through titration experiments, where serial dilutions of the antibody (e.g., 0.125-1.0 μg per test) are tested to identify the concentration that provides maximum separation between positive and negative populations while minimizing background fluorescence. For the H44 clone, 0.25 μg per test has been pre-determined as optimal for peripheral blood cells and granulocytes . To account for autofluorescence and non-specific binding, incorporate fluorescence-minus-one (FMO) controls and matched isotype controls (FITC-conjugated rabbit IgG for polyclonal antibodies or FITC-conjugated mouse IgG for monoclonal antibodies). For absolute quantification, researchers should use calibration beads with known quantities of fluorochrome to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF) or antibody binding capacity (ABC). Data analysis should include careful gating strategies: first on viable cells using a viability dye, then on specific cell populations of interest, and finally analyzing IL18R1 expression within these populations. Report results as percent positive cells and mean/median fluorescence intensity (MFI). When comparing IL18R1 expression across experimental conditions, standardize acquisition settings using quality control beads and perform instrument calibration regularly. Note that cytokine stimulation, particularly with IFN-alpha and IL12, significantly upregulates IL18R1 expression in NK and T cells , so experimental timing and conditions must be consistent across comparisons.

What are the best practices for analyzing co-expression of IL18R1 with other immune markers using multiparameter flow cytometry?

Analyzing co-expression of IL18R1 with other immune markers through multiparameter flow cytometry requires careful panel design and rigorous methodology. Begin by designing a comprehensive antibody panel that includes FITC-conjugated IL18R1 antibodies alongside markers for cell identification (e.g., CD3, CD4, CD8 for T cells; CD56 for NK cells) and functional characterization (e.g., PD-1, CTLA4) . To minimize spectral overlap between fluorophores, place FITC (IL18R1) far from fluorochromes with similar emission profiles such as PE. Implement proper compensation using single-stained controls for each fluorochrome to mathematically correct for spillover. When analyzing data, employ a hierarchical gating strategy: first identify viable cells, then major cell populations, and finally analyze IL18R1 co-expression with other markers within these populations. For Th1 cells, which prominently express IL18R1, examine co-expression with T-bet and IFN-γ. For NK cells, analyze IL18R1 in conjunction with functional markers like perforin, granzyme B, and activation markers. Utilize visualization tools such as bivariate plots, histograms, and heatmaps to represent co-expression patterns. To detect correlations between IL18R1 and other markers, calculate Spearman's correlation coefficients. Be aware that stimulation with IL12 enhances IL18R1 expression on T cells and B cells , potentially altering co-expression patterns. For statistical robustness, analyze sufficient events (minimum 50,000 total events, with at least 100 events in rare populations) and perform replicate experiments. When comparing across disease states or experimental conditions, use dimension reduction techniques like tSNE or UMAP to identify novel co-expression patterns that may not be apparent in conventional bivariate analysis.

How should researchers interpret changes in IL18R1 expression in different experimental conditions?

Interpreting changes in IL18R1 expression across experimental conditions requires consideration of biological context and methodological variables. First, establish a baseline expression profile in your experimental system, noting that IL18R1 is predominantly expressed on Th1 cells, NK cell subsets, neutrophils, and IL-12-stimulated B cells . When interpreting expression changes, consider the kinetics of IL18R1 regulation—IFN-alpha and IL12 are known to induce receptor expression in NK and T cells , with peak expression typically occurring 12-24 hours after stimulation. Changes in IL18R1 expression should be correlated with functional readouts such as cytokine production (particularly IFN-γ and IL6), as IL18R1 expression determines IL18-induced cytokine production from T cells . In disease models, decreased IL18R1 expression may indicate pathological processes; for instance, in lung squamous cell carcinoma, IL18R1 downregulation correlates with poor prognosis and shorter survival . When comparing IL18R1 expression between control and experimental groups, calculate fold changes in mean fluorescence intensity (MFI) rather than relying solely on percentage of positive cells, as this provides more accurate quantification of expression level changes. For Western blot analysis, normalize IL18R1 band intensities to loading controls before calculating fold changes. Be aware that IL18R1 may form complexes with other proteins, such as Na-Cl cotransporter (NCC), which can affect its detection and function . In genetic models (e.g., knockouts or overexpression), confirm the alteration of IL18R1 at both mRNA and protein levels, as post-transcriptional regulation may occur. Finally, consider that IL18R1 functions in a complex with IL18R beta chain for high-affinity IL18 binding ; thus, changes in either component can affect the receptor's functional output.

How can researchers troubleshoot problems with background fluorescence and non-specific binding when using FITC-conjugated IL18R1 antibodies?

Troubleshooting background fluorescence and non-specific binding issues with FITC-conjugated IL18R1 antibodies requires a systematic approach to identify and address specific causes. For high background in flow cytometry applications, first optimize the antibody concentration through careful titration experiments—start with the recommended concentration (e.g., 0.25 μg per test for the H44 clone) and test serial dilutions to determine the optimal signal-to-noise ratio. Implement rigorous blocking protocols using either 2-5% serum from the same species as the secondary antibody or commercial blocking buffers containing irrelevant immunoglobulins. For cells with high Fc receptor expression (especially macrophages and B cells), pre-block with Fc receptor blocking reagents for 15-20 minutes before antibody addition. When analyzing tissue samples, consider autofluorescence sources—implement quenching protocols such as Sudan Black B (0.1-0.3%) treatment for 10-20 minutes or commercial autofluorescence quenchers. For cross-reactivity issues, verify antibody specificity using IL18R1 knockout or knockdown controls. The polyclonal antibody targeting amino acids 21-120 of IL18R1 shows reactivity with human and rabbit samples and predicted reactivity with mouse, rat, dog, cow, and pig , but validation is necessary for each species. In multicolor flow cytometry, perform proper compensation to address spectral overlap between FITC and other fluorophores with similar emission spectra (like PE). For imaging applications, incorporate a washing optimization step—test different detergents (Tween-20, Triton X-100) at various concentrations (0.05-0.3%) and extend washing times to reduce non-specific binding. Finally, if high background persists despite these measures, consider alternative detection strategies, such as using unconjugated primary antibodies with fluorophore-conjugated secondary antibodies for signal amplification and greater specificity.

How does IL18R1 expression correlate with immune cell infiltration in different pathological conditions?

IL18R1 expression demonstrates significant correlations with immune cell infiltration across various pathological conditions, providing important insights into disease mechanisms and potential therapeutic targets. In lung squamous cell carcinoma (LUSC), IL18R1 expression shows robust positive associations with stromal, immune, and estimate scores, indicating its integral role in shaping the tumor microenvironment . Specifically, IL18R1 expression levels correlate strongly with infiltration of T cells, cytotoxic cells, and CD8+ T cells, suggesting its importance in anti-tumor immunity . These correlations extend to key immune cell markers, including CD8A, PD-1, and CTLA4, highlighting potential interactions between IL18R1 signaling and immune checkpoint pathways . In atherosclerosis models, the relationship between IL18R1 and immune infiltration appears more complex. Studies with apolipoprotein E-deficient (Apoe−/−) mice show that the absence of IL18r alone does not significantly affect atherosclerosis development, but combined deficiency of IL18r and Na-Cl cotransporter (NCC) results in more pronounced reductions in plasma inflammatory cytokines like IFN-γ, IL6, and IL18 . This suggests that IL18R1 works in concert with alternative receptors to influence immune cell function in vascular pathology. Flow cytometric analysis with FITC-conjugated IL18R1 antibodies enables precise quantification of receptor expression across different immune cell subsets within pathological tissues. By employing multiparameter approaches, researchers can correlate IL18R1 expression with specific functional states of immune cells, such as activation, exhaustion, or cytokine production profiles. These correlations provide mechanistic insights into how IL18R1 signaling shapes immune responses in different disease contexts and may identify opportunities for therapeutic intervention targeting the IL18/IL18R1 axis to modulate immune cell infiltration and function.

What is the significance of IL18R1 in inflammatory and autoimmune diseases, and how can FITC-conjugated antibodies contribute to research in these areas?

IL18R1 plays a pivotal role in inflammatory and autoimmune diseases through its participation in cytokine signaling networks that orchestrate immune responses. The IL18/IL18R1 axis is crucial for the production of IFN-γ and other pro-inflammatory cytokines that drive pathogenesis in numerous autoimmune conditions. In atherosclerosis, a chronic inflammatory disease, IL18R1 functions in conjunction with the Na-Cl cotransporter (NCC) to mediate IL18-induced cytokine production . While IL18R1 deficiency alone does not significantly impact atherosclerosis in apolipoprotein E-deficient mice, combined deficiency of IL18R1 and NCC leads to substantial reductions in plasma inflammatory cytokines, including IFN-γ, IL6, and IL18 . This suggests that IL18R1 works through redundant pathways in inflammatory disease progression. FITC-conjugated IL18R1 antibodies offer several significant advantages for research in inflammatory and autoimmune conditions. These antibodies enable direct visualization of IL18R1 expression on specific immune cell populations, particularly Th1 cells and NK cells, which are major contributors to autoimmune pathology. This allows researchers to monitor receptor expression changes during disease progression or following therapeutic intervention. In multiparameter flow cytometry applications, FITC-conjugated IL18R1 antibodies can be combined with markers for cell activation, cytokine production, and tissue homing to characterize pathogenic immune cell subsets with high precision. For mechanistic studies, these antibodies facilitate the investigation of receptor internalization, receptor-ligand interactions, and downstream signaling events following IL18 stimulation. Furthermore, in situ imaging with FITC-conjugated antibodies enables the assessment of IL18R1 expression within inflammatory tissue microenvironments, providing spatial context for receptor distribution relative to tissue damage. By correlating IL18R1 expression patterns with clinical parameters and treatment responses, researchers can potentially identify patient subsets who might benefit from therapies targeting the IL18/IL18R1 pathway in conditions ranging from rheumatoid arthritis to inflammatory bowel disease and multiple sclerosis.

What emerging technologies can enhance the application of FITC-conjugated IL18R1 antibodies in single-cell analysis?

Emerging technologies are revolutionizing single-cell applications of FITC-conjugated IL18R1 antibodies, enabling unprecedented insights into receptor dynamics and cellular heterogeneity. Spectral flow cytometry represents a significant advance over conventional flow cytometry by capturing the complete emission spectrum of fluorophores rather than specific bandwidths, allowing better separation of FITC from spectrally similar fluorochromes and enabling more comprehensive immunophenotyping panels that include IL18R1 alongside numerous other markers. Mass cytometry (CyTOF) can be integrated with fluorescence-based detection by using FITC-conjugated IL18R1 antibodies in initial screens to identify populations of interest, which can then be deeply characterized using metal-tagged antibodies against up to 40 additional parameters. Single-cell RNA sequencing combined with protein detection (CITE-seq) allows simultaneous measurement of IL18R1 protein expression (using oligonucleotide-tagged antibodies derived from the same clones used for FITC conjugation) and transcriptomic profiles, revealing relationships between receptor expression and global gene expression patterns. Advanced imaging technologies like imaging flow cytometry combine the quantitative power of flow cytometry with spatial information, allowing researchers to track IL18R1 internalization and colocalization with signaling molecules following ligand binding. Super-resolution microscopy techniques (STORM, PALM, STED) overcome the diffraction limit of conventional microscopy, enabling nanoscale visualization of IL18R1 distribution on the cell membrane and its interaction with other components of the signaling complex, including IL-18R beta chain. Microfluidic systems for single-cell analysis can be coupled with FITC-conjugated IL18R1 antibodies to correlate receptor expression with functional outputs such as cytokine secretion at the individual cell level. These technological advances collectively enhance our ability to understand the complex role of IL18R1 in diverse immune cell populations and disease states with unprecedented resolution and depth.

How might IL18R1-targeted therapies evolve based on current research findings, and what role will FITC-conjugated antibodies play in their development?

The development of IL18R1-targeted therapeutics is poised to advance significantly based on recent research findings, with FITC-conjugated antibodies playing crucial roles in their discovery and validation. Current research highlighting IL18R1's downregulation in lung squamous cell carcinoma and its association with poor prognosis suggests potential for IL18R1 agonist approaches in cancer immunotherapy . Conversely, the involvement of IL18R1 in inflammatory conditions points toward antagonist strategies for autoimmune diseases. FITC-conjugated IL18R1 antibodies will be instrumental throughout the therapeutic development pipeline. In target validation, these antibodies enable precise quantification of receptor expression across diverse tissues and disease states, helping identify conditions where IL18R1-targeted approaches might be most effective. During lead optimization, FITC-conjugated antibodies facilitate high-throughput screening assays to identify compounds that modulate IL18R1 signaling, with fluorescence readouts indicating receptor binding, internalization, or downstream pathway activation. Competitive binding assays using FITC-conjugated antibodies can characterize the binding properties of therapeutic candidates, including binding affinity, epitope specificity, and kinetics. For pharmacodynamic biomarker development, these antibodies can monitor changes in IL18R1 expression or localization following therapeutic intervention, providing crucial feedback on drug efficacy. In patient stratification efforts, FITC-based flow cytometry assays measuring IL18R1 expression might identify individuals most likely to respond to targeted therapies, supporting precision medicine approaches. Recent insights into alternative IL18 receptors, such as the Na-Cl cotransporter (NCC) , suggest that comprehensive targeting of IL18 signaling might require addressing multiple receptors simultaneously. Additionally, emerging understanding of competing endogenous RNA networks involving IL18R1, such as interactions with AC091563.1, RBPMS-AS1, and miR-128-3p , opens avenues for RNA-based therapeutic approaches targeting IL18R1 expression. Throughout these developments, FITC-conjugated IL18R1 antibodies will remain essential tools for mechanistic studies, target engagement assessment, and biomarker development.