EMR3 Antibody

Basic Research Questions

• What is EMR3 and what is its biological significance in immune function?

  EMR3 (EGF module containing, mucin-like hormone receptor-3) is a member of the class B seven-span transmembrane (TM7) receptor family, also classified as an adhesion G protein-coupled receptor (Adhesion G protein-coupled receptor E3). This protein is characterized by an extended extracellular region with N-terminal epidermal growth factor (EGF)-like domains coupled to a TM7 domain via a mucin-like spacer domain .

  EMR3 is predominantly expressed by cells of the immune system and plays a significant role in myeloid-myeloid interactions during immune and inflammatory responses. Unlike other EGF-TM7 family members, EMR3 has a distinctive expression pattern, making it valuable for studying myeloid cell function and differentiation .

• Which immune cell populations express EMR3 and how does its expression pattern differ from other EGF-TM7 family members?

  EMR3 demonstrates a specific expression pattern across myeloid lineage cells:

  Cell Type	EMR3 Expression Level
  Granulocytes	Highest
  Neutrophils	High
  CD16+ (mature) Monocytes	High
  CD16- Monocytes	Dim/Low
  Myeloid Dendritic Cells	Dim/Low
  Macrophages	Positive
  Lymphocytes	Negative
  Plasmacytoid DC	Negative
  CD34+ Hematopoietic Stem Cells	Negative

  A distinguishing characteristic of EMR3 compared to other EGF-TM7 receptors (like CD97 and EMR2) is that EMR3 is not expressed on CD34+CD33−/CD38− committed hematopoietic stem cells or CD34+CD33+/CD38+ progenitors in bone marrow. Instead, EMR3 is specifically upregulated during late stages of granulopoiesis, making it the first EGF-TM7 family member found predominantly on granulocytes .

• What are the principal applications of EMR3 antibodies in immunological research?

  EMR3 antibodies have several key research applications:

  • Flow Cytometry: For identifying and characterizing myeloid cell populations, particularly granulocytes and monocyte subsets. Most protocols recommend using 0.25 µg per 10^6 cells .

  • Immunohistochemistry: For detecting EMR3 in tissue sections, particularly in studies of inflammatory conditions and cancer. Optimal concentrations range from 5-25 µg/mL for paraffin-embedded sections .

  • CyTOF (Mass Cytometry): EMR3 antibodies can be labeled for use in high-dimensional immunophenotyping .

  • Functional Studies: For investigating myeloid cell differentiation and immune responses, including blocking studies to understand EMR3's role in cellular interactions .

  • Cancer Research: Particularly in studying glioblastoma, where EMR3 expression correlates with invasiveness and patient outcomes .

Advanced Research Questions

• How does EMR3 contribute to invasive phenotypes in glioblastoma multiforme and what evidence supports its potential as a therapeutic target?

  EMR3 has been identified as a potential mediator of invasive behavior in glioblastoma multiforme (GBM). Research evidence showing this relationship includes:

  • Microarray analysis through The Cancer Genome Atlas (TCGA) demonstrated a significant survival benefit for GBM patients with tumors showing EMR3 downregulation (P<0.001) .

  • Immunohistochemistry on primary GBM tissue samples revealed variable EMR3 expression patterns: robust expression (>25% of tumor cells) in 37.5% (3/8) of samples, weak expression (1-5% of tumor cells) in 12.5% (1/8), and no detectable expression in 50% (4/8) .

  • siRNA knockdown experiments demonstrated that suppressing EMR3 expression in SF767 glioblastoma cells had a greater than 3-fold reduction in cellular invasion compared to controls (3.42 vs 1, p<0.05), while having no impact on cellular proliferation .

  • Western blot and RT-PCR analysis showed variable EMR3 expression across different glioma cell lines, with SF-767 showing high levels, U87 showing low levels, and U251 and G55 showing no expression .

  These findings suggest that EMR3 could serve as both a prognostic marker and a potential therapeutic target in GBM, particularly for addressing the invasive characteristics that make GBM treatment challenging.

• What methodological approaches have successfully demonstrated EMR3 expression during myeloid cell differentiation?

  Several experimental approaches have been employed to characterize EMR3 expression during myeloid differentiation:

  • In vitro differentiation models: Studies using HL-60 cells (a promyelocytic cell line) and CD34+ progenitor cells have demonstrated that EMR3 is specifically upregulated during late granulopoiesis, unlike other EGF-TM7 receptors that appear earlier in differentiation .

  • Flow cytometric analysis: Using antibodies like clone 908235 or 3D7, researchers have mapped EMR3 expression across the myeloid differentiation spectrum. This approach revealed that EMR3 is absent on early progenitors but increases as cells progress toward terminal differentiation, particularly in the granulocyte lineage .

  • Bone marrow examination: Comparative analysis of bone marrow populations has shown that while CD97 and EMR2 are expressed on CD34+ progenitors, EMR3 is specifically absent on these early cells and appears only in more differentiated stages .

  • Correlation with maturation markers: EMR3 expression has been shown to correlate with the expression of CD16 on monocytes, with more mature CD16+ monocytes expressing higher levels of EMR3 compared to CD16- monocytes .

  These methodological approaches collectively demonstrate that EMR3 serves as a marker for more terminally differentiated myeloid cells, particularly granulocytes.

• What structural features differentiate EMR3 from other EGF-TM7 family members at the protein level?

  EMR3, like other EGF-TM7 receptors, is expressed at the cell surface as a heterodimeric molecule consisting of:

  • A long extracellular α-chain containing EGF-like domains at its N-terminus

  • A membrane-spanning β-chain

  The protein undergoes autocatalytic processing at the G-protein-coupled receptor-proteolytic site (GPS) proximal to the first transmembrane segment, which cleaves the polypeptide into these two subunits that noncovalently associate at the cell surface .

  EMR3's distinguishing features include:

  • Its specific amino acid sequence (human EMR3 spans Met1-Tyr652, Accession # Q9BY15)

  • Its unique expression pattern compared to other family members (CD97, EMR1, EMR2, and EMR4)

  • Its close genetic linkage to the gene encoding EGF-like molecule containing mucin-like hormone receptor 2 on chromosome 19

  While all EGF-TM7 family members share a similar domain organization, their ligand specificity and expression patterns differ significantly, with EMR3 being unique in its predominant expression on mature granulocytes and its absence from hematopoietic stem cells .

Methodological Questions

• What are the optimal protocols for detecting EMR3 via flow cytometry in various cell populations?

  Based on established research protocols, the following methodological approach is recommended for EMR3 detection by flow cytometry:

  Protocol for detection in peripheral blood monocytes:

  1. Sample preparation:

     • Isolate peripheral blood monocytes using standard density gradient separation

     • Wash cells in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin

  2. Staining procedure:

     • Use 0.25 µg of anti-EMR3 antibody (e.g., clone 908235) per 10^6 cells

     • For two-color analysis, co-stain with Mouse Anti-Human CD14 APC-conjugated Monoclonal Antibody (e.g., FAB3832A)

     • Include appropriate isotype controls (e.g., Mouse IgG Flow Cytometry Isotype Control, MAB0041)

     • For indirect staining, use a secondary antibody such as Phycoerythrin-conjugated Anti-Mouse IgG (e.g., F0102B)

     • Alternatively, directly conjugated antibodies can be used (e.g., PE-conjugated Anti-Human EMR3, FAB8496P)

  3. Analysis parameters:

     • Gate on monocyte population based on forward/side scatter characteristics

     • Analyze EMR3 expression in correlation with CD14 to distinguish monocyte subsets

     • Set quadrant markers based on isotype control staining

  For detection in transfected cell lines (validation studies):

  • Use cell lines like HEK293 transfected with human EMR3 and eGFP

  • Include irrelevant transfectants as negative controls

  • Co-expression of eGFP allows for gating on successfully transfected cells

• What tissue preparation methods yield optimal results for EMR3 immunohistochemistry in clinical samples?

  Based on published protocols, the following tissue preparation methods have been successfully employed for EMR3 immunohistochemistry:

  1. Tissue fixation and embedding:

     • Use immersion fixation in formalin

     • Process tissues through standard paraffin embedding protocols

  2. Section preparation:

     • Cut paraffin sections at 4-6 μm thickness

     • Mount sections on positively charged slides

  3. Antigen retrieval (critical step):

     • Perform heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (e.g., CTS013)

     • This step is essential for optimal EMR3 detection in fixed tissues

  4. Staining protocol:

     • Apply Mouse Anti-Human EMR3 Monoclonal Antibody (e.g., MAB8496) at 5 µg/mL

     • Incubate for 1 hour at room temperature

     • Follow with incubation with an appropriate detection system (e.g., Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody, VC001)

     • Develop with DAB (3,3'-diaminobenzidine) chromogen (brown)

     • Counterstain with hematoxylin (blue)

  5. Expected results:

     • In lung cancer tissue, specific staining has been localized to the cytoplasm in cancer cells

     • In GBM, variable expression patterns have been observed across different patient samples

  This protocol has been successfully used to demonstrate EMR3 expression in both normal tissues (for studying myeloid cells) and pathological specimens (particularly in cancer research).

• How should researchers validate the specificity of EMR3 antibodies in experimental systems?

  Comprehensive validation of EMR3 antibodies should include these methodological approaches:

  1. Positive and negative cell line controls:

     • Use cell lines with known EMR3 expression status:

       • SF-767 (high expression)

       • U87 (low expression)

       • U251 and G55 (negative expression)

     • Test antibodies on HEK293 cells transfected with human EMR3 versus irrelevant transfectants

  2. Correlation with transcript levels:

     • Verify protein detection results with RT-PCR analysis

     • Strong correlation between protein and transcript levels supports antibody specificity

  3. Flow cytometry validation:

     • Compare staining on cell populations with known EMR3 expression patterns:

       • Granulocytes (high expression)

       • CD16+ monocytes (high expression)

       • CD16- monocytes (dim/low expression)

       • Lymphocytes (negative)

     • Always include appropriate isotype controls

  4. Blocking and competition studies:

     • Pre-incubate antibodies with recombinant EMR3 protein

     • Observe elimination or reduction of staining

  5. Cross-reactivity assessment:

     • Test antibodies on cells expressing other EGF-TM7 family members

     • Confirm specificity for EMR3 versus CD97, EMR1, EMR2, or EMR4

  6. Multiple antibody comparison:

     • Compare results using antibodies targeting different epitopes of EMR3 (e.g., clone 908235 versus 3D7)

     • Concordant results strengthen validation

  These validation approaches are essential for ensuring reliable and reproducible results in EMR3 research.

• What critical controls should be incorporated in EMR3 antibody-based experimental designs?

  Rigorous experimental design for EMR3 studies should include these controls:

  1. Isotype controls:

     • For flow cytometry: Mouse IgG Flow Cytometry Isotype Control (e.g., MAB0041, IC0041P)

     • Match the isotype to the primary antibody (typically Mouse IgG1 for anti-EMR3)

  2. Cellular controls:

     • Positive controls:

       • Peripheral blood granulocytes (high expression)

       • CD16+ monocytes (high expression)

     • Negative controls:

       • Lymphocytes (no expression)

       • CD34+ hematopoietic stem cells (no expression)

  3. Expression knockdown controls:

     • siRNA knockdown of EMR3 to confirm antibody specificity

     • This approach has been successfully used in SF767 cells

  4. Tissue controls:

     • Known positive tissues (e.g., normal neutrophil-rich tissues)

     • Serial sections with primary antibody omission

  5. Secondary antibody controls:

     • Sections/cells treated with secondary antibody only

  6. Transfection controls for overexpression studies:

     • HEK293 cells transfected with human EMR3 and eGFP

     • Irrelevant transfectants as negative controls

  7. Multiple detection method confirmation:

     • Correlation between different techniques (e.g., flow cytometry, IHC, Western blot)

     • Cross-validation with different antibody clones targeting EMR3

  These controls help ensure the reliability and reproducibility of experimental results involving EMR3 antibodies and minimize the risk of false positive or negative findings.