c3ar1 Antibody

What is C3AR1 and why is it important as a research target?

C3AR1 (C3a anaphylatoxin chemotactic receptor) is a G protein-coupled receptor involved in the complement system that binds to the anaphylatoxin C3a. It consists of 482 amino acids with a molecular weight of approximately 53.9 kDa and contains a uniquely large extracellular domain between transmembrane regions 4 and 5 (over 160 amino acid residues) . C3AR1 has emerged as a critical research target for several reasons:

• It serves as a key mediator in complement-driven inflammatory responses

• It shows selective expression in specific disease states, particularly in NPM1-mutated acute myeloid leukemia (AML) cells compared to normal hematopoietic stem cells

• It engages PTEN (phosphatase and tensin homolog) to negatively regulate neutrophil mobilization, making it relevant for inflammatory pathologies

• It has potential as a therapeutic target due to its differential expression pattern between disease and healthy states

The receptor's involvement in both immune regulation and pathological processes makes C3AR1 antibodies essential tools for investigating these biological pathways.

What types of C3AR1 antibodies are available for research applications?

Researchers can choose from over 480 anti-C3AR1 antibodies from more than 30 different suppliers, with options that vary by several important characteristics :

When selecting a C3AR1 antibody, researchers should consider the following factors:

• Recognition epitope: Some antibodies target the N-terminal domain while others target the large extracellular loop or C-terminal region

• Validation status: Whether the antibody has been validated in your specific application

• Clone specificity: Different monoclonal clones may recognize distinct conformational states of the receptor

• Cross-reactivity: Many antibodies show reactivity across species such as human, mouse, rat, bovine, and even chicken

How can I validate the specificity of my C3AR1 antibody?

Validating antibody specificity is crucial for experimental reliability. For C3AR1 antibodies, consider implementing these validation protocols:

• Positive and negative controls: Use cell lines or primary cells with known C3AR1 expression (myeloid cells as positive; certain lymphoid cells as negative)

• Knockout validation: Test the antibody on C3AR1 knockout or knockdown samples. C3AR1-/- samples should show no detection

• Peptide competition: Pre-incubate the antibody with the immunizing peptide before application to samples

• Multi-antibody concordance: Compare results from antibodies targeting different epitopes of C3AR1

• Correlation with mRNA expression: Compare protein detection with C3AR1 mRNA levels using RT-qPCR

For flow cytometry applications specifically, researchers should compare staining with appropriate isotype controls and evaluate expression patterns across different cell populations, as exemplified in studies of NPM1-mutated AML where C3AR1 shows selective expression patterns .

What are the optimal protocols for detecting C3AR1 using flow cytometry?

Flow cytometry is one of the most common methods for detecting C3AR1, particularly in hematological research. Based on published protocols used in AML research, the following approach is recommended :

• Sample preparation:

  • Isolate mononuclear cells using density gradient centrifugation

  • Wash cells twice in phosphate-buffered saline with 2% fetal bovine serum (PBS/2% FBS)

  • Adjust concentration to 1 × 10^6 cells per 100 μL

• Staining procedure:

  • Block Fc receptors with human serum or commercial Fc block for 10 minutes

  • Add PE-conjugated anti-C3AR1 antibody at manufacturer-recommended concentration (typically 1-5 μg/mL)

  • Include appropriate fluorescence-minus-one (FMO) and isotype controls

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

  • Wash twice with PBS/2% FBS

  • Resuspend in 300-500 μL buffer with viability dye

• Data acquisition considerations:

  • Collect at least 10,000 events in the population of interest

  • Use multiparameter panels to distinguish C3AR1 expression across cell subsets

  • Include CD34, CD38, and other lineage markers for stem/progenitor cell analysis

• Analysis tips:

  • Calculate median fluorescence intensity (MFI) to quantify expression levels

  • Compare to isotype control to establish positive cutoffs

  • Present data as both percentage of positive cells and MFI ratio

How can I optimize Western blot protocols for C3AR1 detection?

Detecting C3AR1 by Western blot can be challenging due to its membrane localization and post-translational modifications. Based on research practices, consider these optimization steps:

• Sample preparation:

  • Use specialized membrane protein extraction buffers containing 1-2% detergents (CHAPS, NP-40, or Triton X-100)

  • Add protease inhibitors to prevent degradation

  • Avoid boiling samples above 70°C to prevent aggregation of membrane proteins

  • Include PNGase F treatment on parallel samples to evaluate glycosylation effects

• Gel electrophoresis and transfer:

  • Use gradient gels (4-12% or 4-15%) for better resolution

  • Transfer at lower voltage for longer periods (25V overnight) for efficient transfer of membrane proteins

  • Use PVDF membrane with 0.45 μm pore size

  • Consider wet transfer methods rather than semi-dry for better results

• Detection optimization:

  • Test multiple anti-C3AR1 antibodies targeting different epitopes

  • Use longer blocking times (2 hours to overnight) with 5% milk or BSA

  • Extended primary antibody incubation (overnight at 4°C) at 1:500 to 1:1000 dilution

  • Include positive control lysates from cells known to express C3AR1 (e.g., activated neutrophils or monocytes)

• Expected results:

  • C3AR1 typically appears around 54 kDa, but glycosylation may result in bands between 60-65 kDa

  • Multiple bands may indicate different glycosylation states

What are optimal immunohistochemistry (IHC) protocols for detecting C3AR1 in tissue samples?

For effective IHC detection of C3AR1 in tissue samples, researchers should consider the following protocol adaptations:

• Tissue preparation:

  • For FFPE (formalin-fixed paraffin-embedded) samples, use heat-induced epitope retrieval with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For frozen sections, fix briefly in cold acetone (10 minutes) or 4% paraformaldehyde (15 minutes)

• Staining optimization:

  • Use amplification systems (e.g., tyramide signal amplification) for low-expression tissues

  • Test both monoclonal and polyclonal antibodies, as tissue fixation may affect epitope accessibility

  • Block endogenous peroxidase activity and include additional blocking for endogenous biotin if using biotin-based detection

  • Incubate primary antibody overnight at 4°C at concentrations of 1:50 to 1:200

• Validation controls:

  • Include tissues with known positive expression (spleen, lung, brain sections)

  • Use inflammation models as positive controls for increased expression

  • Include C3AR1-/- tissue sections as negative controls when available

• Counterstaining considerations:

  • Use light hematoxylin counterstaining to avoid masking specific signals

  • Consider double-staining with lineage markers to identify cell types expressing C3AR1

How can C3AR1 antibodies be used to study NPM1-mutated acute myeloid leukemia?

Research has identified C3AR1 as a selectively expressed marker on NPM1-mutated AML cells, making C3AR1 antibodies valuable tools for studying this disease subtype . Key applications include:

• Diagnostic and prognostic applications:

  • Flow cytometric analysis to differentiate NPM1-mutated from wild-type AML cells

  • Multiparameter analysis combining C3AR1 with GPR56 to identify leukemic stem cells (LSCs) in NPM1-mutated AML

  • Monitoring treatment response by tracking C3AR1-expressing cells

• Therapeutic targeting strategies:

  • Antibody-dependent cellular cytotoxicity (ADCC) assays using anti-C3AR1 antibodies

  • Ex vivo screening of anti-C3AR1 antibodies for NK cell-mediated killing of primary AML cells

  • Development of antibody-drug conjugates targeting C3AR1-expressing cells

• Mechanistic studies:

  • Investigate C3a/C3AR signaling in AML cells through ERK1/2 activation analysis

  • Assess survival pathways in response to C3AR stimulation

  • Evaluate C3AR1 as a therapeutic target by comparing antibody effectiveness against primary AML cells versus normal hematopoietic cells

Research has demonstrated that anti-C3AR1 antibodies efficiently elicit natural killer cell-mediated killing of primary AML cells ex vivo, suggesting potential therapeutic applications .

How can I design experiments to study C3AR1's role in neutrophil mobilization?

C3AR1 plays a critical role in controlling neutrophil mobilization from bone marrow, particularly in inflammatory contexts. To study this function, consider these experimental approaches:

• In vivo mobilization assays:

  • Compare neutrophil counts in blood of wild-type versus C3AR1-/- mice at baseline and after inflammatory stimuli

  • Use bone marrow chimera approaches to distinguish peripheral from central C3AR1 roles

  • Assess neutrophil accumulation in tissues following inflammatory challenges

• Signaling pathway analysis:

  • Investigate PTEN engagement by C3AR1 and subsequent effects on PI3K/AKT pathway

  • Analyze C-X-C chemokine receptor signaling in the presence and absence of C3AR1

  • Measure changes in key neutrophil-mobilizing factors in bone marrow extracellular fluid

• Rescue experiments:

  • Assess whether reinstating C3AR1 expression in C3AR1-/- models through bone marrow transplantation rescues phenotypes

  • Test pharmacological approaches targeting downstream pathways

• Human translational studies:

  • Correlate C3AR1 expression and function with circulating neutrophil numbers in clinical samples

  • Analyze C3AR1 expression patterns in patients with inflammatory conditions

A study on spinal cord injury demonstrated that loss of C3AR1 leads to increased neutrophil mobilization and worsened outcomes, suggesting that targeting C3AR1 may be therapeutically relevant for neutrophil-driven inflammatory conditions .

What are the considerations for using C3AR1 antibodies in therapeutic development?

Developing therapeutic antibodies targeting C3AR1 requires careful consideration of several factors:

• Target specificity and safety profile:

  • Evaluate expression patterns across healthy tissues to minimize off-target effects

  • C3AR1 expression on normal tissues (brain, heart, lung, placenta, spleen, and intestine) must be considered for toxicity risk assessment

  • Monocytes expressing CD16 (approximately 10% of monocytes) show C3AR1 expression and may be affected by therapeutic antibodies

• Antibody design considerations:

  • Epitope selection targeting the large extracellular domain between TM4 and TM5

  • Antibody format selection (IgG1 vs IgG4, full-length vs fragments) based on desired effector functions

  • Fc engineering to enhance or reduce ADCC/CDC activity depending on therapeutic goals

• Efficacy assessment approaches:

  • Ex vivo killing assays with primary patient cells and NK effectors

  • Patient-derived xenograft models to assess in vivo efficacy

  • Combined approaches with standard chemotherapy or other targeted agents

• Biomarker development:

  • Develop companion diagnostics to identify patients with high C3AR1 expression

  • Track C3AR1 expression as a response biomarker during treatment

Research in NPM1-mutated AML has shown promising results with C3AR1-targeting antibodies, including selective killing of leukemic cells while sparing vital hematopoietic stem and progenitor cell populations .

How can I address background issues when using C3AR1 antibodies in flow cytometry?

High background is a common challenge when using C3AR1 antibodies in flow cytometry. These targeted troubleshooting approaches can help:

• Sources of background and solutions:

  • Fc receptor binding: Use dedicated Fc blocking reagents 15-30 minutes before antibody addition

  • Dead cell binding: Include viability dye and exclude dead cells from analysis

  • Non-specific binding: Optimize antibody concentration with titration experiments

  • Autofluorescence: Use spectral compensation and fluorophores that avoid overlap with autofluorescence spectra

• Protocol modifications:

  • Increase washing steps (3-4 times) with cold buffer

  • Reduce primary antibody incubation temperature to 4°C

  • Use filter-sterilized buffers to remove particles

  • Consider adding 1-2% BSA or 10% normal serum from the same species as secondary antibody

• Analytical approaches:

  • Use fluorescence-minus-one (FMO) controls for more accurate gating than isotype controls

  • Establish staining index calculations to optimize signal-to-noise ratio

  • Include biological negative controls (cell types known not to express C3AR1)

What strategies can address inconsistent Western blot results for C3AR1?

C3AR1 detection by Western blot can produce variable results due to its membrane localization and post-translational modifications. Consider these advanced troubleshooting strategies:

• Sample preparation refinements:

  • Test different membrane protein extraction methods (native vs. denaturing conditions)

  • Compare different detergents (digitonin for milder extraction vs. SDS for complete denaturation)

  • Evaluate the impact of sample storage conditions and freeze-thaw cycles

• Technical adaptations:

  • Try alternative blocking agents (commercial protein-free blockers vs. casein vs. BSA)

  • Test different antibody incubation buffers (TBS-T vs. PBS-T with varying detergent concentrations)

  • Consider on-membrane protein refolding protocols for conformation-dependent antibodies

• Post-translational modification considerations:

  • Analyze effects of deglycosylation enzymes (PNGase F, Endo H) on band patterns

  • Investigate phosphorylation status with phosphatase treatments

  • Consider the impact of ubiquitination and other modifications

• Advanced detection methods:

  • Try capillary Western systems for improved sensitivity and reproducibility

  • Consider proximity ligation assays for detecting C3AR1 interactions

  • Use mass spectrometry to confirm antibody specificity

How do I interpret contradictory results between different detection methods for C3AR1?

Researchers may encounter situations where different detection methods yield seemingly contradictory results for C3AR1 expression. This methodological analysis can help reconcile such discrepancies:

• Common causes of discrepancies:

  • Different antibodies may recognize distinct epitopes or conformational states

  • Fixation/processing methods may differentially affect epitope accessibility

  • Detection thresholds vary between methods (flow cytometry vs. IHC vs. Western blot)

  • Cell surface vs. total cellular protein detection differences

• Resolution approaches:

  • Validate using orthogonal methods (e.g., mRNA quantification by RT-qPCR or RNA-seq)

  • Compare multiple antibodies targeting different epitopes

  • Assess detection sensitivity limits for each method

  • Consider the biological context of each sample (activation state, tissue type)

• Method-specific considerations:

  • Flow cytometry detects surface expression on intact cells

  • Western blot represents total protein across the sample

  • IHC provides spatial information but may have lower sensitivity

  • RNA methods detect transcript but not protein levels or localization

• Reconciliation strategies:

  • Develop an integrated interpretation considering the strengths and limitations of each method

  • Design follow-up experiments that specifically address contradictions

  • Consider functional assays to determine biological relevance of expression

What emerging applications of C3AR1 antibodies show promise in basic and translational research?

Several innovative applications of C3AR1 antibodies are emerging in both basic science and translational research:

• Single-cell applications:

  • Integration of C3AR1 detection in mass cytometry (CyTOF) panels for deep immune profiling

  • Single-cell RNA sequencing combined with antibody-based protein detection (CITE-seq) to correlate C3AR1 protein expression with transcriptional states

  • Spatial transcriptomics with antibody detection to map C3AR1+ cells in tissue microenvironments

• Therapeutic development platforms:

  • Bispecific antibody formats targeting C3AR1 and activating immune effectors

  • CAR-T cell development using anti-C3AR1 scFv domains for targeting NPM1-mutated AML

  • ADC (antibody-drug conjugate) approaches leveraging selective C3AR1 expression

• Functional genomics integration:

  • CRISPR-based screening combined with C3AR1 antibody readouts

  • Optogenetic control of C3AR1 signaling pathways

  • Chemical-genetic approaches to selectively modulate C3AR1 function

• Diagnostic applications:

  • Development of antibody-based companion diagnostics for stratifying AML patients

  • Liquid biopsy approaches to detect C3AR1+ circulating tumor cells

Recent research has demonstrated that C3AR1 in combination with GPR56 can distinguish leukemic stem cells in NPM1-mutated AML from normal hematopoietic stem cells, suggesting potential for refined diagnostic and therapeutic targeting approaches .

How can researchers design studies to explore the therapeutic potential of C3AR1 antibodies in inflammatory conditions?

Given C3AR1's role in controlling neutrophil mobilization and inflammatory responses, researchers can design studies to explore therapeutic antibody applications:

• Preclinical model selection and design:

  • Acute inflammatory models: Complement-dependent tissue injury models

  • Chronic inflammation: Autoimmune disease models where neutrophil dysregulation is implicated

  • Comparative studies using genetic knockouts vs. antibody-mediated inhibition

  • Dose-response and timing studies to establish optimal therapeutic windows

• Mechanism of action investigations:

  • Functional blocking vs. depletion approaches

  • Pathway analysis focusing on PTEN and PI3K/AKT signaling

  • Combination approaches with existing anti-inflammatory agents

  • Effects on neutrophil function beyond mobilization (NETosis, phagocytosis, ROS production)

• Translational considerations:

  • Biomarker development to identify patients likely to respond

  • Ex vivo testing of patient samples with C3AR1 antibodies

  • Safety profiling with focus on infection risk and complement system perturbations

  • Development of human-specific or humanized antibodies for clinical translation

• Novel delivery approaches:

  • Tissue-targeted delivery to affected organs

  • Controlled-release formulations for chronic conditions

  • Antibody engineering to modulate half-life and tissue penetration

Research has shown that C3AR1 deficiency leads to increased neutrophil mobilization and worse outcomes in spinal cord injury models, suggesting that therapeutic approaches targeting this receptor could have broad applications in neutrophil-driven inflammatory pathologies .