GPR55 Antibody, FITC conjugated

What is GPR55 and what are its primary functions?

GPR55 (G Protein-Coupled Receptor 55) is a seven transmembrane/G-protein coupled receptor that functions as a receptor for several ligands including L-alpha-lysophosphatidylinositol (LPI) and lysophosphatidylglucoside. This receptor induces calcium (Ca²⁺) release from intracellular stores via the heterotrimeric G protein GNA13 and RHOA signaling pathways. GPR55 has been characterized as a putative cannabinoid receptor and plays significant roles in various physiological processes. It may be involved in hyperalgesia associated with inflammatory and neuropathic pain, and potentially plays a role in bone physiology by regulating osteoclast number and function .

Additionally, recent research has demonstrated that GPR55 activation leads to rapid and transient activation of numerous intracellular signaling pathways. In macrophages, GPR55 acts downstream of lysophosphatidylglucoside to inhibit the translocation of the phospholipid-transporting ABCA1 to plasma membrane and subsequent cholesterol efflux, resulting in lipid accumulation and foam cell formation .

What applications are FITC-conjugated GPR55 antibodies suitable for?

FITC-conjugated GPR55 antibodies are primarily designed for flow cytometry (FCM/FACS) applications, making them ideal for detecting GPR55 expression in various cell populations . Depending on the specific antibody, additional applications may include:

When using these antibodies, it's important to validate the specific application with your sample type, as reactivity may vary between human, mouse, and rat samples depending on the antibody chosen .

What is the difference between various GPR55 antibody immunogen sequences?

Different manufacturers produce GPR55 antibodies targeting distinct amino acid sequences, which may affect specificity and application performance:

The choice of immunogen target is critical as it determines which epitope of the GPR55 protein the antibody recognizes. Antibodies targeting different regions may perform differently in various applications and experimental conditions, particularly if the epitope is affected by protein folding, post-translational modifications, or protein-protein interactions .

How does GPR55 interact with cannabinoid receptor 2 (CB2R) signaling pathways?

Research has revealed a complex interplay between GPR55 and CB2R signaling. GPR55 potently modulates CB2R-mediated responses in neutrophils and other cell types. When GPR55 is activated in human blood neutrophils, it augments the migratory response towards the CB2R agonist 2-arachidonoylglycerol (2-AG), while simultaneously inhibiting neutrophil degranulation and reactive oxygen species (ROS) production .

The molecular mechanism involves interference between GPR55 and CB2R signaling pathways at the level of small GTPases, particularly Rac2 and Cdc42. This interaction ultimately leads to cellular polarization and efficient migration, while abrogating degranulation and ROS formation in neutrophils. Functionally, GPR55 appears to limit tissue-injuring inflammatory responses mediated by CB2R, while synergizing with CB2R in recruiting neutrophils to inflammation sites . This dual regulatory role suggests GPR55 as a potential therapeutic target for inflammation-related conditions.

What methodological considerations are important when using FITC-conjugated GPR55 antibodies for flow cytometry?

When using FITC-conjugated GPR55 antibodies for flow cytometry, several key considerations should be addressed:

• Antibody titration: Optimal dilution ranges typically fall between 1:20-100 for flow cytometry applications. Researchers should perform titration experiments to determine the optimal concentration for their specific cell type .

• Permeabilization protocol: Since GPR55 is found predominantly intracellularly in some cell types (including neutrophils and HL60 cells), proper permeabilization is essential for detection . Standard permeabilization protocols using 0.1% Triton X-100 or commercial permeabilization buffers are recommended.

• Controls: Include appropriate isotype controls (FITC-conjugated rabbit IgG) and positive controls (HEK-GPR55 transfected cells) to validate staining specificity .

• FITC spectral considerations: FITC has excitation/emission peaks at 495/519 nm, which may overlap with other common fluorophores. Proper compensation should be performed when multiplexing with other fluorescent antibodies.

• Light sensitivity: FITC is susceptible to photobleaching, so samples should be protected from light during processing and analysis.

How can I validate GPR55 antibody specificity in my experimental system?

Validating antibody specificity is critical for reliable research outcomes. For GPR55 FITC-conjugated antibodies, a multi-faceted approach is recommended:

• Positive and negative control cells: Use cell lines with confirmed GPR55 expression (such as HEK-GPR55 transfected cells) as positive controls and non-transfected HEK293 cells as negative controls .

• Western blot verification: Confirm the antibody detects a band of the expected size (~37 kDa for GPR55) in positive control samples while showing no bands in negative controls .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (e.g., the peptide sequence from Human G-protein coupled receptor 55 protein AA 203-222) to block specific binding sites. This should eliminate specific staining if the antibody is truly specific .

• Gene silencing: Use siRNA or CRISPR-Cas9 to knock down GPR55 in your experimental system and confirm reduced staining intensity.

• Multiple antibody concordance: Compare results using antibodies targeting different epitopes of GPR55 (e.g., AA 203-222 vs. AA 141-240). Consistent results across different antibodies increase confidence in specificity .

What are the signaling mechanisms through which GPR55 regulates inflammatory responses?

GPR55 regulation of inflammatory responses involves several interconnected signaling pathways:

• GPR55-mediated Ca²⁺ signaling: Upon activation by ligands such as L-alpha-lysophosphatidylinositol (LPI), GPR55 induces calcium release from intracellular stores. This process is mediated through the heterotrimeric G protein GNA13 and RHOA signaling cascade .

• Small GTPase regulation: GPR55 interferes with CB2R signaling at the level of small GTPases, such as Rac2 and Cdc42. This interaction affects cellular polarization, migration, degranulation, and ROS production in neutrophils .

• ERK pathway modulation: GPR55 activation leads to transient activation of ERK signaling, contributing to morphological changes including cell rounding and stress fiber formation .

• ABCA1 translocation inhibition: In macrophages, GPR55 activation downstream of lysophosphatidylglucoside inhibits the translocation of the phospholipid-transporting ABCA1 to the plasma membrane. This inhibition reduces cholesterol efflux, resulting in lipid accumulation and foam cell formation—a key process in inflammatory cardiovascular conditions .

Understanding these signaling mechanisms provides potential targets for therapeutic intervention in inflammatory conditions and suggests experimental approaches for investigating GPR55 function in different cellular contexts.

What is the optimal protocol for detecting GPR55 in neutrophils using FITC-conjugated antibodies?

For optimal detection of GPR55 in neutrophils using FITC-conjugated antibodies, the following protocol is recommended:

• Sample preparation:

  • Isolate neutrophils from fresh human blood using standard density gradient centrifugation methods

  • Resuspend cells at 1×10⁶ cells/mL in PBS with 2% FBS

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization:

  • Since GPR55 is predominantly found intracellularly in neutrophils, permeabilize cells with 0.1% Triton X-100 for 10 minutes at room temperature

  • Wash twice with PBS containing 2% FBS

• Antibody staining:

  • Block non-specific binding with 5% normal goat serum for 30 minutes

  • Incubate cells with FITC-conjugated anti-GPR55 antibody at a dilution of 1:50 (for bs-7686R-FITC) or according to manufacturer's recommendation for 45-60 minutes at room temperature in the dark

  • Wash three times with PBS containing 2% FBS

• Controls and analysis:

  • Include a FITC-conjugated isotype control (e.g., FITC-conjugated rabbit IgG)

  • Analyze by flow cytometry using appropriate FITC detection settings (excitation 488 nm, emission 530/30 nm)

  • Use HEK-GPR55 cells as positive controls and non-transfected HEK293 cells as negative controls

This protocol accounts for the intracellular localization of GPR55 in neutrophils and optimizes detection sensitivity while minimizing background staining.

How should FITC-conjugated GPR55 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of FITC-conjugated GPR55 antibodies is critical for maintaining their performance over time:

• Storage temperature:

  • Store at -20°C or -80°C as recommended by the manufacturer

  • Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• Buffer conditions:

  • Most GPR55 FITC-conjugated antibodies are supplied in buffers containing:

    • 50% Glycerol

    • 0.01M PBS or TBS (pH 7.4)

    • 0.03% Proclin 300 as a preservative

    • Sometimes 1% BSA for stability

  • Do not alter the buffer composition unless specifically required

• Light exposure:

  • FITC is sensitive to photobleaching

  • Keep antibodies protected from light during storage and handling

  • Use amber tubes for storage or wrap containers in aluminum foil

• Working dilutions:

  • Prepare working dilutions immediately before use

  • Do not store diluted antibody solutions for extended periods

• Shipping and temporary storage:

  • If temporary storage above freezing temperatures is necessary, keep at 4°C and protected from light

  • Return to -20°C for long-term storage as soon as possible

Following these guidelines will help maintain antibody performance and extend shelf life, ensuring reliable experimental results over time.

What experimental design approaches are recommended for studying GPR55-CB2R interactions?

To effectively study GPR55-CB2R interactions, a comprehensive experimental design approach is recommended:

• Cell model selection:

  • Primary human neutrophils for physiological relevance

  • HL60 cells (undifferentiated or differentiated) as a more accessible model

  • HEK293 cell lines stably expressing:

    • CB2R alone (HEK-CB2R)

    • GPR55 alone (HEK-GPR55)

    • Both receptors (HEK-CB2R/GPR55)

• Receptor expression verification:

  • Western blot analysis using specific antibodies against GPR55 (~37 kDa) and CB2R (~45 kDa)

  • Immunofluorescence to confirm receptor localization

  • Flow cytometry to quantify receptor expression levels

• Functional assays:

  • Migration assays using 2-arachidonoylglycerol (2-AG) as CB2R agonist

  • ROS production assays to measure respiratory burst

  • Degranulation assays measuring release of specific granule markers

  • Ca²⁺ mobilization assays using fluorescent calcium indicators

• Signaling pathway analysis:

  • Small GTPase activity assays for Rac2 and Cdc42

  • Pull-down assays to assess activation states

  • Co-immunoprecipitation to detect protein-protein interactions

  • Phosphorylation status of downstream targets using phospho-specific antibodies

• Pharmacological interventions:

  • Selective agonists and antagonists for both GPR55 and CB2R

  • Pathway inhibitors to dissect specific signaling components

  • Dose-response studies to determine optimal concentrations

This multifaceted approach enables comprehensive characterization of GPR55-CB2R interactions and their functional consequences in immune cell responses.

What are common issues when detecting GPR55 using FITC-conjugated antibodies and how can they be resolved?

These troubleshooting approaches address the most common challenges researchers face when working with FITC-conjugated GPR55 antibodies and provide practical solutions to improve experimental outcomes .

How can researchers address specificity concerns when studying GPR55 in systems with cannabinoid receptor expression?

When studying GPR55 in systems that also express cannabinoid receptors, addressing specificity concerns requires several strategic approaches:

• Genetic manipulation strategies:

  • Use CRISPR-Cas9 to generate GPR55 knockout cells while maintaining CB2R expression

  • Create cell lines with controlled expression of either or both receptors

  • Use siRNA for selective knockdown of GPR55 or CB2R to distinguish their functions

• Pharmacological approaches:

  • Utilize GPR55-selective ligands that do not activate CB2R

  • Apply CB2R-selective compounds with no activity at GPR55

  • Use antagonists to selectively block either receptor while activating the other

  • Include appropriate vehicle controls for all compounds

• Analytical considerations:

  • Perform receptor binding assays to confirm ligand selectivity

  • Conduct concentration-response studies to identify potential off-target effects

  • Use both overlapping and distinct readouts for GPR55 and CB2R activation

  • Include appropriate genetic controls (receptor-null cells) in all experiments

• Alternative detection methods:

  • Use epitope-tagged receptors to distinguish between GPR55 and CB2R

  • Apply proximity ligation assays to study potential receptor interactions

  • Consider receptor dimerization studies to assess physical interactions

These approaches help distinguish GPR55-specific effects from those mediated by cannabinoid receptors, enabling more precise characterization of GPR55 function in complex biological systems.

What are emerging applications for GPR55 antibodies in disease-related research?

The unique interaction between GPR55 and CB2R suggests several promising research directions for GPR55 antibodies:

• Inflammatory disorders:

  • Investigation of GPR55 expression in tissues from patients with chronic inflammatory conditions

  • Correlation of GPR55 levels with disease severity and inflammatory markers

  • Development of GPR55-targeted therapies for conditions like rheumatoid arthritis and inflammatory bowel disease

• Pain management research:

  • Exploration of GPR55's role in hyperalgesia associated with inflammatory and neuropathic pain

  • Identification of GPR55 expression patterns in pain-processing neural pathways

  • Development of dual GPR55/CB2R modulators as novel analgesics

• Cardiovascular disease:

  • Analysis of GPR55's role in macrophage foam cell formation in atherosclerosis

  • Investigation of GPR55-mediated inhibition of ABCA1 translocation and cholesterol efflux

  • Exploration of GPR55 as a therapeutic target for atherosclerosis and related conditions

• Bone disorders:

  • Study of GPR55's role in regulating osteoclast number and function

  • Investigation of GPR55 expression in osteoporosis and other bone pathologies

  • Development of GPR55 modulators for treating bone density disorders

These emerging applications highlight the potential of GPR55 antibodies as valuable tools for understanding disease mechanisms and developing targeted therapeutic approaches.

How can researchers optimize multiparameter flow cytometry incorporating GPR55 FITC antibodies?

Optimizing multiparameter flow cytometry with GPR55 FITC antibodies requires careful consideration of several technical aspects:

• Panel design considerations:

  • Choose complementary fluorophores that minimize spectral overlap with FITC (avoid PE, Alexa Fluor 488)

  • Pair FITC-conjugated GPR55 antibodies with fluorophores like APC, PE-Cy7, or BV421 for other markers

  • Allocate FITC to GPR55 if expression is expected to be low, as FITC has moderate brightness

• Compensation strategy:

  • Prepare single-color controls for each fluorophore in your panel

  • Use compensation beads for consistent signal intensity

  • Consider automated compensation algorithms but verify results manually

  • Include fluorescence minus one (FMO) controls to set accurate gates

• Sample preparation optimization:

  • Standardize fixation and permeabilization protocols to maintain consistent GPR55 staining

  • If co-staining for membrane and intracellular markers, consider sequential staining protocols

  • Optimize staining buffer composition to reduce background and non-specific binding

  • Titrate all antibodies individually before combining in multicolor panels

• Analysis approach:

  • Implement hierarchical gating strategies starting with forward/side scatter to isolate intact cells

  • Use bivariate plots to analyze GPR55 expression in conjunction with other markers

  • Consider dimensionality reduction techniques (tSNE, UMAP) for complex datasets

  • Apply standardized analysis templates for consistency across experiments