S1PR2 Antibody, Biotin conjugated

What is S1PR2 and why is it important in biological research?

S1PR2 (also known as S1P2 or EDG5) is one of five G-protein-coupled receptors for sphingosine-1-phosphate (S1P), a biologically active metabolic product of sphingolipid. S1PR2 mediates pleiotropic functions encompassing cell proliferation, survival, and migration. It plays critical regulatory roles in normal physiology and disease processes, particularly in the immune, central nervous, and cardiovascular systems . Research has demonstrated S1PR2's significant role in various cancers, where it generally exhibits anti-tumor functions. For example, S1PR2 negatively regulates migration and invasion of human melanoma, glioblastoma, oral squamous cell carcinoma, and gastric cell lines, and inhibits cell proliferation in human renal tumor cells . The receptor's expression is significantly altered in colorectal cancer, with complete loss in 33% of cases and significant decrease in 55%, making it a valuable research target for understanding cancer progression mechanisms .

What are the primary applications for S1PR2 antibodies in research?

S1PR2 antibodies are versatile research tools used in multiple experimental applications. Based on published literature, these antibodies have been successfully employed in:

• Western Blot (WB): For protein detection and quantification (43 publications)

• Immunohistochemistry (IHC): For tissue localization (9 publications)

• Immunofluorescence (IF): For cellular localization (12 publications)

• Immunoprecipitation (IP): For protein complex isolation (2 publications)

• ELISA: For quantitative protein detection

Additionally, S1PR2 antibodies are essential for investigating knockout/knockdown models, with 8 publications specifically utilizing these antibodies in KD/KO studies . When selecting an S1PR2 antibody, researchers should consider the specific application needs and validate reactivity with their target species (human, mouse, rat) as documented in published research.

What is the molecular structure and expected detection pattern for S1PR2?

The protein is encoded by the gene ID 9294 (NCBI) and corresponds to GenBank accession number BC069598 and UniProt ID O95136 . Understanding these specific molecular characteristics is crucial when designing experiments to detect and analyze S1PR2, as different detection methods may reveal distinct patterns based on protein conformation, modifications, or interactions with other cellular components.

How should I design experiments to investigate S1PR2 expression changes in cancer versus normal tissue?

When investigating S1PR2 expression changes between cancer and normal tissues, a multi-methodological approach is recommended based on published research protocols. One effective strategy, as implemented in colorectal cancer research, includes:

• Sample collection and preparation: Work with matched specimens (cancer tissue and adjacent normal tissue from the same patients). In published studies, researchers analyzed 76 pairs of human CRC and matched normal mucosa specimens .

• Immunohistochemistry protocol:

  • Use validated anti-S1PR2 antibodies (for example, anti-mouse/human rabbit S1PR2 antibody, Acris, diluted 1:200)

  • Incubate for 1 hour at room temperature

  • For detection, employ systems like MACH 1 Universal HRP-Polymer and Betazoid DAB Chromogen Kit

• Comparative analysis: Quantify expression levels using established scoring systems. In CRC research, expression patterns were categorized as:

  • Complete loss (observed in 33% of cases)

  • Significantly decreased (observed in 55% of cases)

  • Comparable to normal mucosa (observed in only 12% of cases)

• Controls and validation: Include both positive and negative controls, and consider validating findings with alternative methods such as qPCR or Western blotting for protein quantification.

What controls should be included when using biotin-conjugated S1PR2 antibodies in immunofluorescence studies?

When conducting immunofluorescence studies with biotin-conjugated S1PR2 antibodies, a comprehensive control strategy is essential:

• Primary controls:

  • Positive tissue control: Use tissues known to express S1PR2 (e.g., specific regions of the brain, immune tissues, or cell lines with confirmed expression like HeLa, Daudi, HuH-7, or MCF-7)

  • Negative tissue control: Use tissues from S1PR2 knockout models or tissues known not to express S1PR2

  • Isotype control: Include rabbit IgG control antibody at the same concentration as the primary antibody to assess non-specific binding

• Secondary controls:

  • Secondary-only control: Omit primary antibody but include streptavidin-fluorophore to assess background from secondary detection

  • Autofluorescence control: Process tissue without any antibodies to identify intrinsic tissue fluorescence

  • Blocking control: Test the effectiveness of your blocking protocol by comparing blocked versus non-blocked samples

• Specificity controls:

  • Peptide competition: Pre-incubate the biotin-conjugated S1PR2 antibody with excess S1PR2 immunogen peptide to confirm binding specificity

  • Cross-reactivity assessment: Test the antibody on cells overexpressing related receptors (S1PR1, S1PR3, etc.) to confirm specificity

• Technical controls:

  • Concentration gradient: Test a range of antibody dilutions (e.g., 1:100, 1:200, 1:500) to determine optimal signal-to-noise ratio

  • Counterstaining: Include nuclear staining (DAPI/Hoechst) and possibly membrane markers to properly contextualize S1PR2 localization

These controls should be systematically documented and included in methodology sections of publications to ensure experimental rigor.

How can I design experiments to study the functional role of S1PR2 in immune cell recruitment and inflammatory responses?

To investigate S1PR2's role in immune cell recruitment and inflammatory responses, a multi-faceted experimental approach is recommended:

• In vivo models:

  • Compare wild-type (S1PR2+/+) and S1PR2-deficient (S1PR2-/-) mice in inflammation-driven disease models (as demonstrated in colitis-induced cancer models)

  • Use lineage-specific conditional knockout models to distinguish between epithelial and immune cell-specific S1PR2 functions

  • Consider using reporter systems like S1pr2-CreERT2.Rosa26Lox-Stop-Lox-tdTomato mice for fate-mapping studies

• Ex vivo approaches:

  • Isolate and culture organoids from S1PR2+/+ intestines and treat with S1PR2 antagonists (e.g., JTE013) to mimic S1PR2 ablation

  • Use flow cytometry to assess immune cell populations, particularly focusing on:

    • CD4+ T cell proliferation (S1PR2 has been shown to regulate epithelial barrier by preventing CD4+ T-cell proliferation)

    • Other relevant immune cell subsets (CD45+ populations, dendritic cells, macrophages)

• Molecular analysis:

  • Evaluate downstream signaling pathways affected by S1PR2, particularly:

    • AKT phosphorylation (decreased with S1PR2 overexpression)

    • PTEN levels (increased with S1PR2 overexpression)

    • STAT3 activation pathways (S1PR2 signaling is implicated in STAT3 activation)

• Tissue analysis:

  • Perform immunohistochemistry with markers for:

    • Epithelial barrier integrity

    • Immune cell infiltration (CD45+)

    • Cell proliferation (Ki67)

    • Apoptosis (Caspase-3)

Design experiments to include both acute and chronic timepoints, as S1PR2's role may differ between initial inflammatory responses and chronic inflammation settings.

What are the optimal protocols for using biotin-conjugated S1PR2 antibodies in various applications?

Based on published research methodologies, here are optimized protocols for biotin-conjugated S1PR2 antibody applications:

Western Blotting Protocol:

• Sample preparation: Lyse cells in RIPA buffer with protease/phosphatase inhibitors

• Protein separation: Load 20-50μg protein on 10-12% SDS-PAGE gels

• Transfer: Use PVDF membrane (recommended over nitrocellulose for S1PR2)

• Blocking: 5% non-fat milk in TBST, 1 hour at room temperature

• Primary antibody: Dilute biotin-conjugated S1PR2 antibody 1:500-1:2000 in blocking buffer

• Incubation: Overnight at 4°C with gentle rocking

• Detection: Use streptavidin-HRP (1:5000) for 1 hour at room temperature

• Expected band: 40-50 kDa region

Immunohistochemistry Protocol:

• Sample preparation: Fix tissues in 10% neutral buffered formalin, embed in paraffin

• Sectioning: 4-5μm thick sections

• Antigen retrieval: Citrate buffer (pH 6.0), 95°C for 20 minutes

• Blocking: 3% hydrogen peroxide (10 min) + serum blocking (1 hour)

• Primary antibody: Biotin-conjugated S1PR2 antibody (1:200) in Da Vinci Diluent for 1-2 hours at room temperature

• Detection: Streptavidin-HRP and DAB chromogen

• Counterstain: Hematoxylin for nuclear visualization

Immunofluorescence Protocol:

• Cell preparation: Fix with 4% paraformaldehyde (15 min), permeabilize with 0.1% Triton X-100 (10 min)

• Blocking: 5% BSA in PBS, 1 hour at room temperature

• Primary antibody: Biotin-conjugated S1PR2 antibody (1:100-1:400)

• Incubation: 2 hours at room temperature or overnight at 4°C

• Detection: Streptavidin-conjugated fluorophore (Alexa Fluor 488/594/647) at 1:500 dilution

• Counterstain: DAPI (1:1000) for nuclear visualization

• Mounting: Anti-fade mounting medium

Always include appropriate positive controls (HeLa, Daudi, HuH-7, or MCF-7 cells) and negative controls in each experiment to validate results.

How should I optimize antibody concentration for detecting S1PR2 in cells with varying expression levels?

Optimizing antibody concentration is crucial for reliable detection of S1PR2, especially when working with samples of varying expression levels. Based on research methodologies, here's a systematic optimization approach:

Step 1: Establish baseline expression levels
First, determine the expected S1PR2 expression in your experimental system through literature review. Research has established that:

• S1PR2 expression varies significantly across tissues and disease states

• In colorectal cancer, expression can range from complete loss (33% of cases) to levels comparable with normal mucosa (12% of cases)

• Cell lines such as HeLa, Daudi, HuH-7, and MCF-7 have detectable S1PR2 expression and can serve as positive controls

Step 2: Antibody titration matrix
Create a titration matrix testing multiple variables:

For Western blotting applications, the recommended starting range is 1:500-1:2000 , but optimal dilution should be determined experimentally for each specific application and sample type.

Step 3: Signal-to-noise optimization
For each condition, calculate signal-to-noise ratio by comparing specific signal to background. The optimal antibody concentration provides maximum specific signal with minimal background across all expression levels.

Step 4: Validation with controls

• Positive controls: Use samples with confirmed S1PR2 expression (e.g., normal tissues or recommended cell lines)

• Negative controls: Use S1PR2 knockout samples or cells with confirmed absence of S1PR2

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

Step 5: Documentation
Document the optimization process thoroughly, including images of representative results at different antibody concentrations, to guide future experiments and ensure reproducibility.

What are the best fixation and permeabilization methods for preserving S1PR2 epitopes in tissue sections?

Preserving S1PR2 epitopes during fixation and permeabilization is critical for accurate immunodetection. Based on successful protocols in published research, here are optimized methods:

Fixation Options and Outcomes:

Permeabilization Methods:
For membrane proteins like S1PR2, permeabilization must be carefully controlled:

• For paraffin sections:

  • After deparaffinization, perform heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

  • This approach was successfully employed in studies examining S1PR2 expression in colorectal cancer specimens

• For frozen sections:

  • Brief (5-minute) treatment with 0.1% Triton X-100 in PBS

  • Alternatively, 0.1% saponin (milder detergent) for more sensitive epitopes

• For cultured cells:

  • 0.1% Triton X-100 in PBS for 10 minutes at room temperature

  • 0.5% saponin for 5-10 minutes for selective membrane permeabilization

  • Digitonin (25 μg/ml) for 5 minutes for selective plasma membrane permeabilization while preserving internal membranes

Critical considerations:

• S1PR2 is a multi-pass membrane protein, making membrane preservation crucial

• Overfixation can mask epitopes, particularly for biotin-conjugated antibodies

• Extended antigen retrieval may be necessary for formalin-fixed tissues (>20 minutes)

• For dual staining, optimize fixation conditions that work for both targets

• Always validate new fixation/permeabilization methods with known positive controls

Studies examining S1PR2 in tissue specimens have successfully employed these methods, achieving specific staining patterns that correlate with functional data from S1PR2+/+ and S1PR2-/- models .

How should I quantify and interpret S1PR2 staining patterns in different cellular compartments?

Accurate quantification and interpretation of S1PR2 staining patterns requires systematic analysis of compartment-specific expression. Based on research methodologies, here's a comprehensive approach:

Quantification Methods by Cellular Compartment:

• Membrane localization (primary expected location for S1PR2):

  • Use membrane segmentation algorithms or membrane-specific masks

  • Quantify as percentage of membrane showing positive staining

  • Measure intensity along membrane perimeter using line-scan analysis

  • Expected pattern: Distinct membrane staining with potential clustering in signaling domains

• Cytoplasmic localization (may indicate receptor internalization):

  • Measure mean cytoplasmic intensity after excluding nucleus and membrane

  • Calculate cytoplasmic-to-membrane ratio to assess receptor trafficking

  • Expected pattern: Punctate staining may indicate vesicular trafficking

• Nuclear localization (less common but possible):

  • Calculate nuclear-to-cytoplasmic ratio

  • Assess colocalization with nuclear markers (DAPI)

  • Expected pattern: Usually minimal for S1PR2, significant nuclear staining may require validation

Scoring Systems:
For clinical or comparative studies, implement a structured scoring system:

Differential Pattern Analysis:
Research has shown that S1PR2 expression patterns may vary by:

• Cell type (epithelial vs. immune cells)

• Disease state (normal tissue vs. cancer)

• Microenvironmental context (inflammatory vs. normal conditions)

For example, in colorectal cancer research, S1PR2 expression was completely lost in 33% of cases and significantly decreased in 55%, with only 12% retaining expression comparable to normal mucosa . This indicates that loss of membrane expression may be particularly relevant in cancer progression.

Validation Approaches:

• Confirm localization patterns with multiple antibodies targeting different epitopes

• Validate with subcellular fractionation followed by Western blotting

• Use super-resolution microscopy for detailed localization studies

• Include controls from S1PR2-/- tissues to confirm specificity

By applying these quantification approaches, researchers can generate reproducible data that accurately reflects the biological significance of S1PR2 localization patterns.

How do I reconcile contradictory results between S1PR2 antibody detection methods and functional assays?

Reconciling contradictory results between antibody detection and functional assays is a common challenge in S1PR2 research. A systematic troubleshooting approach based on research findings can help resolve these discrepancies:

Common Contradictions and Resolution Strategies:

• Antibody detection shows S1PR2 presence but no functional response

  • Potential causes:

    • Detection of non-functional receptor variants or fragments

    • Post-translational modifications affecting function but not detection

    • Decoupling from downstream signaling pathways

  • Resolution strategies:

    • Use antibodies targeting different epitopes, especially functional domains

    • Perform phosphorylation assays for downstream effectors (AKT, PTEN)

    • Assess receptor coupling to G-proteins with GTPγS binding assays

• Functional S1PR2 responses without detectable antibody signal

  • Potential causes:

    • Low receptor expression below antibody detection threshold

    • Epitope masking by interacting proteins or conformational changes

    • Cross-reactivity with other S1P receptors in functional assays

  • Resolution strategies:

    • Use more sensitive detection methods (amplification systems, super-resolution microscopy)

    • Test alternative antibodies targeting different epitopes

    • Use specific S1PR2 antagonists (JTE013) to confirm functional specificity

• Conflicting results between in vitro and in vivo studies

  • Potential causes:

    • Different microenvironmental contexts affecting receptor expression or function

    • Compensatory mechanisms in vivo not present in vitro

    • Cell type-specific differences in signaling pathways

  • Resolution strategies:

    • Use organoid cultures to bridge in vitro and in vivo contexts

    • Employ tissue-specific or inducible knockout models

    • Compare results across multiple experimental systems

Methodological Reconciliation Framework:

How can I differentiate between specific S1PR2 staining and potential cross-reactivity with other S1P receptors?

Differentiating between specific S1PR2 staining and cross-reactivity with other S1P receptors (S1PR1, S1PR3, S1PR4, S1PR5) requires rigorous controls and validation approaches. Based on research methodologies, here's a comprehensive strategy:

Cross-Reactivity Assessment Framework:

• Sequence homology analysis

  • S1P receptors share significant sequence homology, with the highest conservation in transmembrane domains

  • Identify unique epitopes for S1PR2 by sequence alignment analysis

  • Verify that your S1PR2 antibody targets regions with minimal homology to other S1PRs

• Expression system controls

  • Use cells overexpressing individual S1P receptors:

    • Transfected cell lines expressing single S1PR subtypes

    • siRNA knockdown of S1PR2 to confirm signal reduction

    • Cells from S1PR2-/- mice as negative controls

• Pharmacological validation

  • Employ receptor-specific antagonists:

    • JTE013 for S1PR2 (used in organoid studies)

    • W146 for S1PR1

    • TY52156 for S1PR3

  • Pre-treatment should selectively reduce staining of the targeted receptor

• Comparative staining patterns

  • Different S1P receptors have distinct tissue distribution patterns:

    • S1PR1: Ubiquitous, especially in endothelium and lymphocytes

    • S1PR2: Widespread but prominent in brain, heart, and immune cells

    • S1PR3: Cardiovascular system, lung, kidney

    • S1PR4: Predominantly in lymphoid tissues

    • S1PR5: Brain, skin, natural killer cells

Validation Experiments:

Example from research: Studies investigating S1PR2's role in colorectal cancer employed S1PR2-/- mouse models as definitive controls to confirm antibody specificity. Additionally, they complemented antibody-based detection with functional studies using JTE013 antagonist treatment in organoids to mimic S1PR2 ablation, providing converging evidence of specificity .

What are common issues when using biotin-conjugated S1PR2 antibodies and how can they be resolved?

Researchers frequently encounter specific challenges when working with biotin-conjugated S1PR2 antibodies. Based on research experiences, here are systematic troubleshooting approaches:

Common Issues and Resolution Strategies:

• High background signal

  • Potential causes:

    • Endogenous biotin in tissues (particularly in kidney, liver, brain)

    • Insufficient blocking

    • Non-specific binding of detection reagents

  • Solutions:

    • Implement endogenous biotin blocking using avidin/biotin blocking kits

    • Increase blocking stringency (5-10% serum from same species as secondary + 1% BSA)

    • Include 0.1-0.3% Triton X-100 in blocking solution

    • Use streptavidin-conjugates from different suppliers

• Weak or absent S1PR2 signal

  • Potential causes:

    • Low S1PR2 expression (common in certain cancers, with 33% showing complete loss)

    • Epitope masking due to fixation

    • Biotin accessibility issues

  • Solutions:

    • Implement amplification systems (tyramide signal amplification)

    • Optimize antigen retrieval (extend citrate buffer treatment to 30 minutes)

    • Try alternative fixation methods (acetone for frozen sections)

    • Decrease washing stringency (reduce Tween-20 concentration to 0.05%)

• Non-specific or unexpected staining patterns

  • Potential causes:

    • Cross-reactivity with other S1P receptors

    • Detection of processed/degraded receptor forms

    • Off-target binding

  • Solutions:

    • Validate with tissues from S1PR2-/- mice

    • Compare with non-biotinylated S1PR2 antibodies

    • Perform peptide competition assays

    • Use receptor antagonist (JTE013) treatment as functional validation

• Inconsistent results between experiments

  • Potential causes:

    • Antibody degradation or biotin oxidation

    • Variable S1PR2 expression between samples

    • Inconsistent protocol execution

  • Solutions:

    • Aliquot antibodies to avoid freeze-thaw cycles

    • Include standard positive controls in each experiment

    • Standardize all protocol steps (timing, temperatures, reagent lots)

    • Consider automated staining platforms for consistency

Systematic Troubleshooting Workflow:

• Control validation: Always run parallel positive controls (tissues known to express S1PR2) and negative controls (S1PR2-/- tissues or isotype controls)

• Antibody validation matrix:

• Tissue-specific optimization: Different tissues may require tailored protocols based on endogenous biotin levels and S1PR2 expression patterns

• Documentation: Maintain detailed records of all optimization steps and successful parameters to ensure reproducibility

Research on S1PR2 in colorectal cancer successfully navigated these challenges by implementing rigorous controls and protocol optimization, achieving specific staining that correlated with functional outcomes in S1PR2-/- models .

How can I address inconsistent S1PR2 detection results between different experimental batches?

Addressing batch-to-batch variability in S1PR2 detection requires systematic identification of variability sources and implementation of standardization procedures. Based on research practices, here's a comprehensive approach:

Root Cause Analysis for Batch Inconsistency:

• Antibody factors

  • Potential issues:

    • Lot-to-lot variability in commercial antibodies

    • Degradation of biotin conjugation over time

    • Freeze-thaw cycles affecting antibody performance

  • Solutions:

    • Purchase larger lots and aliquot upon receipt

    • Document lot numbers and correlate with performance

    • Store at -80°C in single-use aliquots with stabilizers

    • Test new lots side-by-side with previous lots before full implementation

• Sample preparation variability

  • Potential issues:

    • Inconsistent fixation duration or penetration

    • Variable antigen retrieval efficiency

    • Tissue thickness differences

  • Solutions:

    • Standardize fixation protocols (time, temperature, solution preparation)

    • Implement consistent tissue processing protocols

    • Consider automated antigen retrieval systems

    • Process all experimental groups simultaneously when possible

• Protocol execution inconsistencies

  • Potential issues:

    • Timing variations between steps

    • Temperature fluctuations

    • Reagent preparation differences

  • Solutions:

    • Create detailed step-by-step protocols with timing specifications

    • Use timers for critical steps

    • Prepare master mixes for multiple samples

    • Consider automated staining platforms

• Biological variability

  • Potential issues:

    • S1PR2 expression varies significantly even within similar samples (33% of CRC show complete loss)

    • Age, treatment conditions, or microenvironment differences

  • Solutions:

    • Increase biological replicates

    • Include standardized positive control samples in each batch

    • Normalize to housekeeping proteins consistently expressed across samples

Standardization Implementation Strategy:

Quality Control Measures:

• Implement a multi-level QC system:

  • Level 1: Technical replicate consistency check within each batch

  • Level 2: Control sample performance evaluation

  • Level 3: Cross-batch calibration using standard samples

• Establish acceptance criteria:

  • Signal-to-noise ratio thresholds

  • Coefficient of variation limits between technical replicates

  • Positive control signal intensity ranges

• Data normalization approaches:

  • Normalize to housekeeping proteins run in parallel

  • Use internal reference standards

  • Consider employing a normalization algorithm that accounts for batch effects

In published research examining S1PR2 expression in colorectal cancer, investigators implemented these standardization approaches to ensure reliable detection across 76 paired tumor and normal samples, enabling them to confidently categorize expression patterns into distinct groups (complete loss, significantly decreased, and normal expression) .

What strategies can resolve epitope masking issues in fixed tissues when detecting S1PR2?

Epitope masking is a significant challenge in detecting membrane proteins like S1PR2 in fixed tissues. Based on research methodologies, here are comprehensive strategies to overcome this challenge:

Antigen Retrieval Optimization:

• Heat-induced epitope retrieval (HIER) methods:

• Proteolytic enzyme retrieval approaches:

  • Proteinase K (10-20 μg/ml, 10-15 min at 37°C)

  • Trypsin (0.05%, 10-20 min at 37°C)

  • Pepsin (0.05%, 5-15 min at 37°C)

  • Note: Enzymatic approaches should be carefully titrated as overdigestion can destroy epitopes

• Combined approaches:

  • Sequential application of HIER followed by mild enzymatic treatment

  • Pressure cooker methods (higher temperature, shorter time)

  • Variable pH approach (test multiple pH conditions)

Fixation Optimization Strategies:

• Alternative fixation methods to reduce epitope masking:

  • Acetone fixation (10 min, -20°C) for frozen sections

  • Methanol-acetone mixture (1:1, 10 min, -20°C)

  • PAXgene or molecular fixatives that preserve protein conformation

  • Zinc-based fixatives that cause less protein cross-linking

• Post-fixation treatments:

  • Sodium borohydride treatment (0.5% in PBS, 10 min) to break methylene bridges

  • Sudan Black B (0.1-0.3%) to reduce both autofluorescence and improve epitope accessibility

  • Glycine treatment (100mM, 10 min) to block free aldehydes

Blocking and Permeabilization Optimization:

• Enhanced blocking strategies:

  • Use mixture of 5% normal serum + 1% BSA + 0.1% cold fish skin gelatin

  • Include 0.1% Triton X-100 in blocking buffer for balanced permeabilization

  • Add 0.1% Tween-20 to reduce non-specific binding

• Stepwise permeabilization:

  • Gentle detergent gradient (start with 0.01% and increase if needed)

  • Freeze-thaw permeabilization for difficult samples

  • Saponin (0.01-0.05%) for more selective membrane permeabilization

Detection Enhancement Approaches:

• Signal amplification systems:

  • Tyramide signal amplification (TSA) for biotin-conjugated antibodies

  • Polymer-based detection systems (more sensitive than ABC method)

  • Quantum dot conjugates for improved sensitivity and stability

• Alternative detection strategies:

  • Multi-antibody approach (use two different S1PR2 antibodies targeting distinct epitopes)

  • Proximity ligation assay for detecting protein interactions involving S1PR2

  • RNAscope to correlate protein detection with mRNA localization

Research investigating S1PR2 in colorectal cancer specimens successfully employed citrate buffer-based heat-induced epitope retrieval followed by carefully optimized immunodetection protocols to achieve specific staining that correlated with functional outcomes in comparative models .

How can I design experiments to investigate the interaction between S1PR2 and downstream signaling pathways in primary immune cells?

Investigating S1PR2-mediated signaling in primary immune cells requires specialized approaches that account for both receptor biology and immune cell characteristics. Based on published research methodologies, here's a comprehensive experimental design framework:

Experimental Approach Overview:

• Cell isolation and characterization:

  • Isolate primary immune cell populations of interest (T cells, B cells, dendritic cells)

  • Flow cytometry characterization of baseline S1PR2 expression levels

  • Compare with S1PR2 expression in established cell lines (calibration controls)

• Modulation of S1PR2 expression/function:

• Key signaling pathways to evaluate:

  • AKT/PTEN axis (S1PR2 overexpression reduces AKT phosphorylation and increases PTEN levels)

  • STAT3 activation (S1PR2 signaling is implicated in STAT3 activation)

  • Rho/ROCK pathway activation (canonical S1PR2 signaling)

  • Cytoskeletal rearrangement (important for immune cell function)

• Functional readouts:

  • Migration assays (S1PR2 negatively regulates migration in multiple cell types)

  • Proliferation assessments (e.g., Ki67 staining, as performed in CRC studies)

  • Cytokine production profiles (important for immune function)

  • Cell-cell interaction dynamics using live imaging

Specific Experimental Protocols:

• S1PR2-AKT/PTEN pathway analysis:

  • Stimulate cells with S1P (10-100nM) with/without JTE013 pre-treatment

  • Harvest cells at multiple timepoints (5, 15, 30, 60 minutes)

  • Assess AKT phosphorylation status (Ser473, Thr308) by Western blot

  • Quantify PTEN levels and phosphorylation state

  • Include controls with PI3K inhibitors (e.g., LY294002) to distinguish pathway-specific effects

• Immune-specific functional studies:

  • T cell studies: Assess impact on CD4+ T cell proliferation (S1PR2 has been shown to regulate epithelial barrier by preventing CD4+ T-cell proliferation)

  • B cell studies: Investigate germinal center (GC) dynamics using S1pr2-CreERT2.Rosa26Lox-Stop-Lox-tdTomato fate-mapping approaches

  • Integrate with in vivo models: Use adoptive transfer of S1PR2-modified cells into appropriate disease models

• Advanced signaling analysis:

  • Phosphoproteomics to identify novel S1PR2-regulated phosphorylation events

  • Proximity labeling (BioID, APEX) to identify S1PR2 interaction partners

  • Single-cell approaches to address heterogeneity in primary immune populations

Integration with in vivo models:

• Adoptive transfer studies with modified cells

• Bone marrow chimeras to distinguish intrinsic vs. extrinsic effects

• Conditional knockout models (e.g., using S1pr2-CreERT2 systems)

Research investigating S1PR2 in various disease models has benefited from these integrated approaches, revealing context-specific roles in different cell types and disease states .

What experimental approaches can elucidate the role of S1PR2 in the tumor microenvironment beyond cancer cells themselves?

Investigating S1PR2's role in the tumor microenvironment (TME) requires approaches that capture complex intercellular interactions. Based on research methodologies, here's a comprehensive experimental framework:

Multi-dimensional Experimental Strategy:

• Spatial characterization of S1PR2 in the TME:

  • Multiplex immunohistochemistry/immunofluorescence:

    • Co-stain S1PR2 with markers for:

      • Cancer cells (e.g., cytokeratin)

      • Immune cell populations (CD45, CD4, CD8, CD68)

      • Endothelial cells (CD31)

      • Fibroblasts (αSMA, FAP)

    • Include markers for cell proliferation (Ki67) and apoptosis (Caspase-3) as done in CRC studies

  • Single-cell spatial transcriptomics:

    • Map S1PR2 expression patterns across different TME niches

    • Correlate with inflammatory and immunosuppressive signatures

• Cell type-specific S1PR2 functions:

• Advanced 3D model systems:

  • Tumor organoid co-culture models:

    • Develop co-cultures of tumor organoids with immune cells

    • Compare S1PR2+/+ vs. S1PR2-/- organoids as established in published research

    • Add specific TME components (fibroblasts, endothelial cells) systematically

    • Treat with S1PR2 antagonist JTE013 to mimic receptor ablation

  • Ex vivo tissue slice cultures:

    • Maintain spatial organization of native TME

    • Short-term drug treatments with S1PR2 modulators

    • Live imaging of immune cell trafficking

• In vivo approaches with TME focus:

  • Cell type-specific knockout models:

    • Use tissue-specific Cre drivers (CD45-Cre, LysM-Cre, Tie2-Cre, etc.)

    • Compare with global S1PR2-/- phenotypes in cancer models

  • Bone marrow chimeras:

    • Transplant S1PR2-/- bone marrow into wild-type recipients (and vice versa)

    • Distinguish between stromal and hematopoietic S1PR2 functions

  • Therapeutic interventions:

    • S1PR2 antagonists in combination with immune checkpoint inhibitors

    • Timeline analysis to distinguish early vs. late effects on TME composition

• Mechanistic analysis of intercellular communication:

  • Exosome/secretome analysis:

    • Compare secreted factors from S1PR2+/+ vs. S1PR2-/- cells

    • Functional testing of conditioned media on recipient cells

  • Cytokine/chemokine profiling:

    • Multiplex analysis of inflammatory mediators

    • Correlation with immune infiltration patterns

Research on colorectal cancer has demonstrated that S1PR2 has distinct roles in different cell types within the TME. For example, studies showed that S1PR2 loss impaired mucosal regeneration and promoted the expansion of intestinal stem cells, with effects on both epithelial and immune cell functions .

How can advanced microscopy techniques be combined with S1PR2 antibodies to reveal receptor dynamics and molecular interactions?

Advanced microscopy techniques offer powerful approaches to elucidate S1PR2 dynamics and interactions at unprecedented resolution. Based on current research methodologies, here's a comprehensive framework for applying these techniques with S1PR2 antibodies:

Super-Resolution Microscopy Approaches:

• Stimulated Emission Depletion (STED) Microscopy:

  • Application for S1PR2: Resolve nanoscale distribution patterns of S1PR2 in membrane microdomains

  • Implementation strategy:

    • Use biotin-conjugated S1PR2 primary antibody with STED-compatible streptavidin-conjugated fluorophores (ATTO647N, Abberior STAR RED)

    • Combine with lipid raft markers (CTxB) to assess compartmentalization

    • Resolution capability: 30-80nm laterally, enabling visualization of receptor nanoclusters

• Single Molecule Localization Microscopy (PALM/STORM):

  • Application for S1PR2: Track individual receptor molecules with nanometer precision

  • Implementation strategy:

    • Use photoswitchable fluorophores conjugated to anti-S1PR2 antibodies

    • Perform dual-color STORM with downstream signaling molecules (G-proteins, PTEN)

    • Quantify clustering patterns and co-localization at single-molecule level

    • Resolution capability: 10-30nm, revealing individual receptor distribution

• Structured Illumination Microscopy (SIM):

  • Application for S1PR2: Visualize dynamic receptor redistribution during signaling

  • Implementation strategy:

    • Compatible with live-cell imaging using anti-S1PR2 Fab fragments

    • Combine with fluorescent sphingosine-1-phosphate analogs to visualize ligand-receptor interactions

    • Resolution capability: 100-120nm, doubling conventional microscopy resolution

Live Cell Imaging Technologies:

• Single Particle Tracking:

  • Application for S1PR2: Analyze receptor diffusion dynamics and confinement

  • Implementation strategy:

    • Label S1PR2 with Quantum Dots via biotin-streptavidin linkage

    • Track receptor movement before and after S1P stimulation

    • Calculate diffusion coefficients in different membrane regions

    • Analyze effect of JTE013 antagonist on mobility patterns

• Förster Resonance Energy Transfer (FRET):

  • Application for S1PR2: Detect molecular interactions with signaling partners

  • Implementation strategy:

    • Create S1PR2-fluorescent protein fusions (careful validation required)

    • Pair with fluorescently tagged interaction partners (G-proteins, PTEN, AKT)

    • Measure FRET efficiency changes following receptor activation

    • Perform acceptor photobleaching FRET for quantitative interaction analysis

• Fluorescence Recovery After Photobleaching (FRAP):

  • Application for S1PR2: Measure receptor lateral mobility and membrane anchoring

  • Implementation strategy:

    • Express S1PR2-GFP constructs at physiological levels

    • Perform targeted photobleaching of membrane regions

    • Compare recovery kinetics in different cell types relevant to CRC research

    • Analyze effect of cytoskeletal disruption on mobility

Correlative and Multi-modal Approaches:

• Correlative Light and Electron Microscopy (CLEM):

  • Application for S1PR2: Connect fluorescence patterns with ultrastructural context

  • Implementation strategy:

    • Label S1PR2 with both fluorescent tags and electron-dense markers

    • Overlay fluorescence data with EM images of the same region

    • Resolve receptor localization relative to cellular ultrastructure

    • Particularly valuable for endosomal trafficking studies

• Expansion Microscopy:

  • Application for S1PR2: Physical expansion of specimens for improved resolution

  • Implementation strategy:

    • Label S1PR2 with conventional antibodies before expansion

    • Achieve 70-100nm resolution with standard confocal microscopy

    • Valuable for thick tissue sections from tumor samples

• Lattice Light-Sheet Microscopy:

  • Application for S1PR2: Rapid 3D imaging with minimal phototoxicity

  • Implementation strategy:

    • Live imaging of S1PR2-GFP in physiologically relevant 3D cultures

    • Track receptor dynamics during cell migration and division

    • Combine with optogenetic approaches for precise spatiotemporal control

Experimental Validation Approaches:

• Use S1PR2-/- tissues or cells as negative controls for antibody specificity

• Implement genetic knockdown/knockout validation alongside advanced imaging

• Perform biological function correlation with imaging findings

Research on receptor dynamics in cancer models has benefited from these advanced techniques, enabling researchers to connect molecular-scale receptor behavior with tissue-level phenotypes observed in models like S1PR2-/- mice .