GPR174 Antibody, FITC conjugated

What is GPR174 and what physiological processes does it regulate?

GPR174 is a G protein-coupled receptor that signals primarily via Gαs in immune cells. Research demonstrates that GPR174 significantly impacts B cell gene expression, controlling a gene expression program that includes CD86 and other immune regulatory molecules . In B cells, GPR174 signaling influences approximately 1,000 genes after just 4 hours of unstimulated culture, affecting critical pathways including the induction of CD86, Nr4a1, and Ccr7 while downregulating ITIM-containing receptors . Beyond B cells, GPR174 also plays important roles in dendritic cell function and maturation, with knockout studies revealing its involvement in inflammatory bowel disease pathogenesis through regulation of dendritic cell-mediated immune responses .

What specific region of GPR174 does the FITC-conjugated antibody target?

The FITC-conjugated GPR174 antibody targets amino acids 283-310 located in the C-terminal region of human GPR174 . This antibody is generated from rabbits immunized with a KLH-conjugated synthetic peptide corresponding to this amino acid sequence. The C-terminal region targeted by this antibody represents an important cytoplasmic domain of the GPR174 receptor, which is critical for downstream signaling events. This specificity makes the antibody particularly useful for detecting intact GPR174 protein in various experimental applications .

What are the primary applications for GPR174 FITC-conjugated antibodies?

FITC-conjugated GPR174 antibodies are valuable research tools primarily used in:

• Flow cytometry: For detecting and quantifying GPR174 expression on cell surfaces, particularly in immune cells like B cells where GPR174 signaling affects activation markers including CD86, CD69, and CD83 .

• Immunofluorescence microscopy: For visualizing GPR174 localization in tissues and cells.

• Western blotting: For detecting GPR174 protein in cell or tissue lysates to evaluate expression levels .

• ELISA: For quantitative measurement of GPR174 in research samples .

The FITC conjugation provides direct fluorescent detection capability without requiring secondary antibodies, which is particularly advantageous in multicolor flow cytometry experiments when studying complex immune cell populations.

How should samples be prepared for optimal GPR174 detection with FITC-conjugated antibodies?

For optimal GPR174 detection with FITC-conjugated antibodies, sample preparation should include:

• For cell suspensions (flow cytometry):

  • Harvest cells gently using non-enzymatic methods when possible to preserve surface epitopes

  • Wash cells in phosphate-buffered saline (PBS) containing 1-2% bovine serum albumin (BSA)

  • Fix cells using 2-4% paraformaldehyde if intracellular staining is required

  • For intracellular staining, permeabilize with 0.1% saponin or 0.1% Triton X-100

• For tissue sections (immunofluorescence):

  • Use freshly frozen or paraformaldehyde-fixed, paraffin-embedded sections

  • Perform antigen retrieval if using fixed tissues

  • Block with appropriate serum (5-10% normal serum) to reduce non-specific binding

• For Western blotting:

  • Extract proteins using RIPA buffer containing protease inhibitors

  • Separate proteins by SDS-PAGE under reducing conditions

  • Transfer to PVDF or nitrocellulose membranes

All procedures should be performed while protecting samples from excessive light exposure to prevent photobleaching of the FITC fluorophore.

How can GPR174 FITC-antibodies be used to investigate the role of GPR174 in B cell activation?

GPR174 FITC-antibodies offer sophisticated approaches to investigate B cell activation mechanisms:

• Dual-parameter flow cytometry: Researchers can simultaneously measure GPR174 expression levels and activation markers like CD86, CD69, and CD83 in B cells under various stimulation conditions, such as BCR ligation with anti-IgM . This allows correlation between GPR174 expression and activation status.

• Time-course experiments: By analyzing GPR174 expression at multiple timepoints (0h, 4h, 24h) following B cell isolation, researchers can track how receptor expression changes during culture-induced activation. Research shows that B cells undergo massive changes in gene expression after just 4 hours in culture, with many changes being GPR174-dependent .

• Comparative analysis of wildtype vs. GPR174-deficient B cells: Using the antibody to confirm absence of GPR174 in knockout models while simultaneously assessing activation markers can reveal the signaling pathways dependent on GPR174.

• Signaling pathway investigation: Combined with intracellular phospho-protein staining, GPR174 surface expression can be correlated with downstream Gαs-mediated signaling events, helping to elucidate the complete signaling cascade in B cell activation .

• Co-localization studies: Using confocal microscopy with multiple fluorophores, researchers can visualize the spatial relationship between GPR174 and other signaling molecules during B cell activation.

What are the critical controls needed when using GPR174 FITC-conjugated antibodies in flow cytometry?

When using GPR174 FITC-conjugated antibodies in flow cytometry, the following controls are essential:

• Isotype control: A FITC-conjugated rabbit IgG that has the same isotype but does not specifically bind to GPR174 must be used at the same concentration to assess non-specific binding .

• Fluorescence-minus-one (FMO) control: Include all antibodies in your panel except the GPR174 FITC antibody to establish proper gating strategies and account for spectral overlap.

• Biological negative control: Cells known to lack GPR174 expression or GPR174 knockout cells should be used to confirm antibody specificity. Studies with GPR174-deficient B cells or dendritic cells provide appropriate negative controls .

• Biological positive control: Cells known to express high levels of GPR174, such as follicular B cells, should be included to verify detection sensitivity .

• Compensation controls: Single-stained controls for each fluorophore in your panel to correct for spectral overlap, particularly important when FITC emission overlaps with other green-yellow fluorophores.

• Unstained control: Completely unstained cells to establish autofluorescence baseline.

• Blocking validation: Compare staining with and without Fc receptor blocking to ensure specific binding to GPR174 rather than through Fc receptors.

These controls ensure reliable and interpretable data when studying GPR174 expression in complex immune cell populations.

How can GPR174 antibodies be used to investigate the relationship between GPR174 and inflammatory bowel disease (IBD)?

GPR174 antibodies can be instrumental in investigating the relationship between GPR174 and IBD through multiple experimental approaches:

• Tissue expression analysis: FITC-conjugated GPR174 antibodies can be used for immunofluorescence microscopy to compare receptor expression in colonic tissue from healthy controls versus IBD patients. Research in mouse models shows that GPR174 knockout alleviates DSS-induced colitis, suggesting altered expression may influence disease progression .

• Dendritic cell characterization: Flow cytometric analysis using GPR174 antibodies can assess expression levels on dendritic cells from intestinal lamina propria. Studies show GPR174 knockout influences CD11c+ dendritic cell infiltration and maturation in DSS-induced colitis, with decreased expression of MHC-II, CD80, and CD86 .

• Ex vivo tissue culture studies: Colonic biopsies can be cultured with or without GPR174 antagonists, and antibodies can be used to monitor receptor expression alongside inflammatory markers. Research indicates GPR174-deficient mice show decreased levels of pro-inflammatory cytokines TNF-α and IL-6, with increased anti-inflammatory IL-10 .

• Barrier function correlation: GPR174 expression levels detected by antibodies can be correlated with intestinal permeability markers. GPR174 knockout mice demonstrate higher expression of tight junction proteins Zo-1 and Occludin, suggesting a role in barrier maintenance .

• T cell differentiation studies: GPR174 antibodies can be used in co-culture experiments with dendritic cells and T cells to understand how receptor expression influences T cell polarization, as GPR174-deficient BMDCs induce more Treg cells and fewer Th1 cells .

This data from rodent models provides a framework for human IBD investigations .

What technical factors affect the sensitivity and specificity of GPR174 FITC-antibody detection?

Several technical factors can significantly impact the sensitivity and specificity of GPR174 FITC-antibody detection:

• Fixation effects: Excessive paraformaldehyde fixation can mask epitopes, particularly those in the C-terminal region (AA 283-310) targeted by this antibody . Optimize fixation time and concentration (typically 2-4% PFA for 10-15 minutes) to balance cell morphology preservation with epitope accessibility.

• Buffer composition: The presence of detergents in staining buffers can affect membrane integrity and accessibility of transmembrane receptors like GPR174. For surface staining, use detergent-free buffers, while intracellular staining may require 0.1% saponin or similar gentle permeabilization agents.

• Antibody concentration: Titration experiments are essential, as both insufficient and excessive antibody concentrations can diminish signal-to-noise ratio. The polyclonal nature of this antibody means batch-to-batch variation may necessitate individual titration .

• Sample handling: GPR174 expression in B cells changes dramatically even during short-term culture, with approximately 1,000 genes showing altered expression after just 4 hours . Minimize time between sample collection and staining to capture accurate in vivo expression levels.

• FITC photobleaching: FITC is particularly susceptible to photobleaching. Protect samples from light during all processing steps and minimize exposure during acquisition, especially for quantitative analyses.

• Blocking protocol: Effective blocking with appropriate serum (typically 5-10% normal serum matching the host species of the secondary antibody) is critical to reduce background signal, particularly in tissues with high autofluorescence like intestinal samples used in IBD studies .

• Flow cytometer settings: Proper PMT voltage settings and compensation are crucial for accurate detection, especially when FITC is used in multicolor panels where spectral overlap is common.

• Antigen retrieval: For fixed tissue samples, optimized antigen retrieval methods may be necessary to expose the C-terminal epitope (AA 283-310) recognized by this antibody .

How should researchers design experiments to investigate GPR174 signaling pathways using FITC-conjugated antibodies?

When designing experiments to investigate GPR174 signaling pathways using FITC-conjugated antibodies, researchers should consider:

• Temporal analysis: GPR174 signaling via Gαs rapidly induces changes in gene expression. Design time-course experiments (0h, 1h, 4h, 24h) to capture both immediate and delayed signaling events, as research shows significant changes occur within 4 hours in B cells .

• Pathway inhibitor approach: Combine GPR174 antibody staining with specific inhibitors of downstream pathways:

  • cAMP pathway inhibitors (for Gαs-mediated effects)

  • PKA inhibitors (H-89 or KT5720)

  • Adenylyl cyclase inhibitors (SQ22536)

  • Phosphodiesterase inhibitors (IBMX)

• Phospho-flow cytometry: Integrate GPR174 surface staining with intracellular phospho-protein detection to correlate receptor expression with activation of downstream effectors such as CREB, which is phosphorylated following Gαs activation.

• Genetic models: Include GPR174-deficient cells as negative controls and for comparative signaling analysis. Studies show strong overlap in gene expression changes between GPR174- and Gαs-deficient B cells, confirming the receptor signals through this pathway .

• Receptor antagonist studies: Compare signaling in the presence and absence of GPR174 antagonists to validate pathway specificity and potentially identify therapeutic targets, as GPR174 antagonists may reduce gene expression shifts and augment B cell survival during culture .

• Co-immunoprecipitation followed by Western blotting: Use GPR174 antibodies to pull down receptor complexes and analyze associated proteins to map signaling networks.

• Functional readouts: Correlate receptor expression with functional outcomes such as:

  • B cell viability and activation marker expression

  • Dendritic cell maturation status (MHC-II, CD80, CD86 expression)

  • T cell stimulatory capacity

This comprehensive approach allows mapping of the complete GPR174 signaling cascade from receptor to functional outcome.

What are the considerations for multiplexing GPR174 FITC-antibodies with other immune cell markers?

When multiplexing GPR174 FITC-antibodies with other immune cell markers, researchers should consider:

• Spectral compatibility: FITC emits in the green spectrum (peak ~520nm), making it potentially problematic to combine with other green fluorophores like GFP or PE. When analyzing complex immune populations, design panels that place other markers in spectrally distinct channels:

  • Far-red/APC for lineage markers (CD19 for B cells, CD11c for dendritic cells)

  • PE-Cy7 or APC-Cy7 for activation markers (CD86, CD80)

  • BV421/Pacific Blue for additional functional markers

• Compensation requirements: FITC has significant spectral overlap with PE, requiring careful compensation. Always include single-stained controls for each fluorophore and perform compensation before analysis.

• Cell-specific analysis strategies: GPR174 expression varies between immune cell types:

  • For B cells: Include CD19, IgD, and CD23 to distinguish follicular B cells (most affected by GPR174 signaling)

  • For dendritic cells: Include CD11c, MHC-II, CD80, and CD86 to assess maturation status alongside GPR174 expression

• Fixation/permeabilization compatibility: If intracellular markers are needed alongside GPR174, ensure the fixation/permeabilization protocol preserves both the GPR174 epitope and other target epitopes. Different fixation protocols may be required for optimal detection of all markers.

• Sequential staining approach: For complex panels, consider staining surface markers first, then fixing/permeabilizing for intracellular markers to minimize epitope masking.

• Panel optimization: When studying GPR174 in the context of IBD, include:

  • Intestinal DC markers (CD11c, MHC-II, CD103, CX3CR1)

  • T cell phenotyping markers (CD4, Foxp3, IFN-γ, IL-10)

  • Epithelial barrier markers if analyzing tissue sections (ZO-1, Occludin)

• Antibody titration: Each antibody in the panel should be individually titrated in the context of the full panel to achieve optimal signal-to-noise ratio without overwhelming compensation limits.

• Dead cell discrimination: Include a viability dye in a far-red channel to exclude dead cells, which can bind antibodies non-specifically.

How should researchers interpret changes in GPR174 expression in different immune cell subpopulations?

When interpreting changes in GPR174 expression across immune cell subpopulations, researchers should consider:

• Baseline expression patterns: Establish normal GPR174 expression levels across immune cell types before interpreting disease-related or experimental changes. GPR174 is expressed at different levels in various B cell subsets and dendritic cell populations .

• Correlation with functional markers: Analyze GPR174 expression in relation to:

  • B cell activation markers (CD86, CD69, CD83)

  • Dendritic cell maturation markers (MHC-II, CD80, CD86)

  • Expression of tight junction proteins in intestinal epithelial cells (ZO-1, Occludin)

• Disease context interpretation: In inflammatory conditions like colitis, changes in GPR174 expression should be interpreted alongside:

  • Local cytokine milieu (TNF-α, IL-6, IL-10 levels)

  • Tissue infiltration patterns of immune cells

  • Clinical disease activity indices

• Signaling pathway context: Changes in GPR174 expression may reflect feedback regulation within the Gαs signaling pathway. Analysis should include assessment of downstream mediators like cAMP levels or CREB phosphorylation .

• Temporal dynamics: GPR174 expression and its effects on gene expression change rapidly over time. Interpret expression data within the specific time frame of the experiment, noting that B cells show significant changes in ~1,000 genes after just 4 hours in culture .

• Population heterogeneity: Use appropriate gating strategies in flow cytometry to distinguish true changes in expression levels from shifts in cell population frequencies, particularly in tissues with complex immune cell compositions like intestinal lamina propria .

• Genetic background effects: Consider strain-specific or genetic background effects when comparing GPR174 expression between different experimental models or patient cohorts, as variants in the GPR174 locus have been associated with autoimmune diseases .

What are potential pitfalls in data interpretation when using GPR174 FITC-antibodies?

Researchers should be aware of several potential pitfalls when interpreting data from GPR174 FITC-antibody experiments:

• Autofluorescence interference: Tissues like intestinal mucosa have significant autofluorescence in the FITC channel, which can be misinterpreted as positive GPR174 staining. Always include unstained and isotype controls, and consider using spectral unmixing techniques for accurate signal separation .

• Ex vivo artifacts: GPR174 signaling affects approximately 1,000 genes in B cells within 4 hours of unstimulated culture, meaning that sample processing time can significantly impact results . Minimize time between tissue collection and analysis, and interpret ex vivo findings with caution.

• Antibody binding specificity: The polyclonal nature of the FITC-conjugated GPR174 antibody means it recognizes multiple epitopes within the C-terminal region (AA 283-310), which may be differentially accessible depending on receptor conformation or protein-protein interactions . Validate key findings using alternative detection methods or antibody clones.

• FITC signal stability: FITC photobleaches relatively quickly compared to other fluorophores. Signal intensity decreases during extended imaging sessions can be misinterpreted as biological phenomena rather than technical artifacts.

• Fixation-induced epitope masking: The C-terminal region targeted by this antibody (AA 283-310) may be differentially accessible depending on fixation protocols . Inconsistent fixation between samples can create artificial differences in detected expression levels.

• Non-specific binding via Fc regions: Insufficient blocking can lead to Fc receptor-mediated binding, particularly in samples rich in B cells, macrophages, or dendritic cells. Always use appropriate Fc receptor blocking and validate with isotype controls .

• Compensatory mechanisms in knockout models: GPR174-deficient mice may develop compensatory signaling mechanisms that complicate interpretation of phenotypes. Changes observed in knockout models should be validated using acute receptor inhibition strategies .

• Context-dependent signaling: GPR174 effects on dendritic cell maturation and T cell differentiation are context-dependent, varying between steady-state and inflammatory conditions . Experimental conditions should closely match the biological context being investigated.

How might GPR174 FITC-antibodies be utilized in studying the therapeutic potential of GPR174 antagonists?

GPR174 FITC-antibodies offer valuable approaches for investigating GPR174 antagonists as potential therapeutics:

• Target engagement studies: Flow cytometry with GPR174 FITC-antibodies can verify whether antagonist compounds effectively bind to GPR174 by measuring changes in antibody binding accessibility. If the antagonist and antibody compete for the same or overlapping binding sites, reduced FITC signal would confirm target engagement.

• Receptor internalization assays: Sequential staining with GPR174 FITC-antibodies before and after antagonist treatment can quantify receptor internalization rates, providing insights into drug mechanism and pharmacodynamics.

• In vivo tracking: Following administration of GPR174 antagonists in experimental IBD models, FITC-conjugated antibodies can be used to monitor receptor expression changes in intestinal tissue, correlating with disease improvement markers. Research suggests GPR174 antagonists may reduce gene expression shifts and augment B cell survival during culture .

• Biomarker development: Flow cytometric analysis of GPR174 expression patterns before and during antagonist therapy could identify patient subgroups most likely to respond to treatment, supporting personalized medicine approaches.

• Mechanistic studies: Combined with phospho-flow cytometry, GPR174 FITC-antibodies can help elucidate how antagonists affect downstream signaling cascades:

  • Changes in cAMP levels (Gαs pathway)

  • Regulation of CD86 and other activation markers

  • Effects on dendritic cell maturation (MHC-II, CD80, CD86 expression)

• Dendritic cell functional assays: GPR174 FITC-antibodies can be used to sort dendritic cell populations based on receptor expression levels, followed by functional assays to assess how antagonists modify:

  • T cell stimulatory capacity

  • Cytokine production profiles

  • Induction of regulatory vs. inflammatory T cell responses

• Safety monitoring: During preclinical and clinical development of GPR174 antagonists, FITC-antibodies could help monitor on-target effects in immune cell populations, providing safety biomarkers for drug development.

What role might GPR174 antibodies play in understanding the link between GPR174 and autoimmune diseases?

GPR174 antibodies can significantly advance our understanding of the link between GPR174 and autoimmune diseases through several research applications:

• Genetic association validation: Variants in the GPR174 locus have been associated with autoimmune diseases . FITC-antibodies can be used to quantify receptor expression levels in patients carrying different genetic variants, establishing functional consequences of these polymorphisms.

• Cell-type specific roles: Flow cytometric analysis with GPR174 FITC-antibodies combined with lineage markers can identify which immune cell populations show altered receptor expression in autoimmune conditions, helping to pinpoint the most relevant therapeutic targets.

• Mechanistic pathway analysis: In autoimmune disease models, GPR174 antibody staining can be combined with:

  • Phospho-flow cytometry to assess downstream signaling

  • Transcriptomic analysis to correlate receptor expression with gene expression changes

  • Functional assays measuring B cell activation and antibody production

• Tissue-specific investigations: Immunofluorescence microscopy using GPR174 antibodies can map receptor expression in affected tissues from autoimmune disease models or patient biopsies, identifying spatial relationships with inflammatory infiltrates.

• Interventional studies: GPR174 antibodies can monitor receptor expression changes during therapeutic interventions in autoimmune disease models, such as the DSS-induced colitis model where GPR174 knockout shows protective effects .

• B cell survival and function: Since GPR174 affects B cell viability and gene expression , antibodies can be used to investigate how receptor signaling contributes to B cell survival, activation, and possibly autoreactive antibody production in autoimmune conditions.

• Dendritic cell-T cell interactions: GPR174 antibodies can help characterize how receptor expression affects dendritic cell maturation and subsequent T cell polarization in autoimmune settings, as GPR174-deficient dendritic cells induce more regulatory T cells and fewer inflammatory Th1 cells .

• Epithelial barrier function: In intestinal autoimmunity, GPR174 antibodies can be used alongside tight junction protein markers to explore how receptor signaling affects barrier integrity, as GPR174 knockout improves intestinal barrier function in colitis models .

How can GPR174 FITC-antibodies be integrated with advanced imaging techniques for deeper insights into receptor biology?

Integration of GPR174 FITC-antibodies with advanced imaging techniques offers powerful approaches for investigating receptor biology:

• Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy with GPR174 FITC-antibodies can reveal nanoscale receptor clustering and co-localization with signaling partners, providing insights into spatial organization beyond diffraction-limited conventional microscopy.

• Live-cell imaging: Using membrane-permeable GPR174 antibody fragments conjugated to FITC enables tracking of receptor dynamics in living cells, revealing:

  • Trafficking patterns during cell activation

  • Internalization kinetics following ligand binding

  • Redistribution during immune synapse formation

• Intravital microscopy: FITC-conjugated GPR174 antibodies can be used for in vivo imaging in mouse models, enabling visualization of receptor expression on immune cells within intact tissues during inflammatory processes such as colitis .

• Correlative light-electron microscopy (CLEM): This technique combines fluorescence imaging of GPR174 FITC-antibodies with electron microscopy, providing both molecular specificity and ultrastructural context to understand receptor localization within cellular compartments.

• Multiplexed ion beam imaging (MIBI): Using metal-tagged GPR174 antibodies alongside dozens of other markers enables highly multiplexed tissue imaging, revealing complex cellular interaction networks in tissues like inflamed intestinal mucosa in IBD models .

• Fluorescence resonance energy transfer (FRET): Combining GPR174 FITC-antibodies (donor) with acceptor fluorophore-labeled antibodies against potential interaction partners can detect molecular proximity below 10nm, revealing direct protein-protein interactions in the signaling pathway.

• Lattice light-sheet microscopy: This technique enables high-speed, low-phototoxicity 3D imaging of GPR174 distribution during dynamic cellular processes such as dendritic cell migration or B cell activation .

• Spectral imaging: Advanced spectral unmixing algorithms can separate FITC signals from tissue autofluorescence, particularly valuable in intestinal tissues where autofluorescence can confound conventional fluorescence imaging in IBD studies .

• Tissue clearing techniques: Methods like CLARITY or iDISCO combined with GPR174 FITC-antibody staining enable whole-organ 3D imaging, revealing global patterns of receptor expression across entire lymphoid organs or intestinal segments.