FOLR2 Antibody, FITC conjugated

What is FOLR2 and why is it important in research?

FOLR2, also known as Folate Receptor beta, is a 38 kDa protein that mediates the cellular uptake of folic acid and reduced folates. It plays crucial roles in key metabolic processes including nucleotide and methionine synthesis, the interconversion of glycine and serine, and histidine breakdown . FOLR2 is predominantly expressed in placenta, neutrophilic lineage cells, and some CD34+ hematopoietic progenitor cells . Its research importance stems from its upregulation in myeloid leukemias, head and neck squamous cell carcinomas, and several non-epithelial cancers . FOLR2 is also significantly upregulated on macrophages and monocytes at chronic inflammatory sites including rheumatoid arthritis synovium and glioblastoma, making it a valuable target for studying inflammation and potential therapeutic interventions .

How does FITC conjugation impact FOLR2 antibody functionality?

FITC conjugation to FOLR2 antibodies occurs via primary amines (lysines) on the antibody structure. Typically, between 3 and 6 FITC molecules are conjugated to each antibody molecule . This conjugation ratio is critical because higher numbers can result in solubility problems and internal quenching that reduces fluorescence brightness . The conjugation process can potentially affect antibody binding if FITC molecules attach near the antigen-binding site. Researchers should validate FITC-conjugated FOLR2 antibodies against unconjugated versions to ensure maintained specificity and affinity. FITC-conjugated antibodies are excited by the 488 nm laser line (typically from an argon laser), with emission collected at approximately 530 nm, making them compatible with standard flow cytometry and fluorescence microscopy setups .

What are the optimal storage conditions for FOLR2 antibody, FITC conjugated?

FOLR2 antibodies with FITC conjugation should be stored at 4°C in the dark to prevent photobleaching of the fluorophore . Long-term storage may require aliquoting to avoid repeated freeze-thaw cycles that can degrade both the antibody and the conjugated fluorophore. PBS is typically used as the storage buffer for FITC-conjugated antibodies . When preparing working solutions, it's advisable to centrifuge the antibody solution briefly before use to remove any aggregates that might have formed during storage. The stability of FITC-conjugated antibodies decreases over time due to photobleaching, so researchers should plan experiments accordingly and verify antibody performance regularly if using stored reagents for extended periods.

How can FOLR2 antibody, FITC conjugated be used to study inflammatory diseases?

FOLR2 is significantly upregulated on macrophages and monocytes at sites of chronic inflammation, making FITC-conjugated FOLR2 antibodies valuable tools for investigating inflammatory conditions . For rheumatoid arthritis research, these antibodies can be used to:

• Identify and quantify FOLR2-expressing macrophages in synovial fluid and tissue using flow cytometry

• Track the trafficking of folate-targeted therapeutic compounds in synovial macrophages

• Monitor changes in FOLR2 expression during disease progression or treatment response

• Investigate FOLR2's role in methotrexate transport in rheumatoid arthritis patients

For multiparameter analysis, researchers can combine FOLR2-FITC with other markers of macrophage activation states using flow cytometry or confocal microscopy. Typical protocols involve incubation of cells with 10 μg/mL of the antibody for 3 hours at room temperature, followed by appropriate washing steps . When analyzing tissue sections, FOLR2-FITC can be counterstained with DAPI to visualize nuclei and assess cellular localization of FOLR2 expression .

What are the current methodologies for investigating FOLR2 in cancer research using FITC-conjugated antibodies?

FOLR2 is upregulated in several cancer types, making FITC-conjugated FOLR2 antibodies important tools in cancer research. Current methodologies include:

For advanced applications, researchers are combining FOLR2-FITC with additional markers to identify specific tumor-associated macrophage populations that may promote cancer progression . The specific subcellular localization of FOLR2 (cell surface and cytoplasm) can provide insights into its functional status within tumor cells and tumor-associated immune cells .

How can I design experiments to investigate the functional relationship between FOLR2 and drug delivery systems?

FOLR2's high affinity for folic acid and its derivatives makes it an attractive target for drug delivery systems, particularly for inflammatory diseases and certain cancers . When designing experiments to investigate this relationship:

• Start with binding assays using FITC-conjugated FOLR2 antibodies to quantify receptor expression levels on target cells and establish baseline measurements.

• Compare receptor-mediated endocytosis of folate-conjugated compounds with and without FOLR2 antibody blocking:

  • Pre-incubate cells with unlabeled anti-FOLR2 antibody

  • Introduce folate-conjugated therapeutic compounds

  • Measure uptake using complementary detection methods

• Use competition assays with varying concentrations of free folate to determine the specificity of drug delivery systems for FOLR2 versus FOLR1 or FOLR3, considering the 68% and 79% sequence homology, respectively .

What is the optimal protocol for detecting FOLR2 using FITC-conjugated antibodies in flow cytometry?

Based on established research protocols, the following methodology is recommended for flow cytometric detection of FOLR2:

• Sample Preparation:

  • For cell suspensions: Harvest cells and adjust to 1×10^6 cells/mL in cold PBS containing 2% FBS

  • For tissue samples: Generate single-cell suspensions through enzymatic digestion with collagenase and DNase I

• Antibody Staining:

  • Block Fc receptors with 10% normal serum for 15 minutes at 4°C

  • Incubate with FOLR2-FITC antibody (1-10 μg/mL depending on manufacturer's recommendation) for 30 minutes at 4°C in the dark

  • Wash cells twice with cold PBS/2% FBS

  • For multicolor panels, include appropriate compensation controls

• Data Acquisition:

  • Excite FITC with 488 nm laser

  • Collect emission at 530 nm (typically FL1 channel)

  • Analyze minimum of 10,000 events per sample

• Analysis Considerations:

  • Use isotype-matched FITC-conjugated control antibodies at the same concentration

  • Include FMO (Fluorescence Minus One) controls for multicolor panels

  • FOLR2 expression typically appears as a shift in fluorescence intensity rather than discrete positive/negative populations

For neutrophils specifically, FOLR2 staining has been demonstrated to localize to cell surfaces and cytoplasm, requiring permeabilization if intracellular staining is desired .

How do I troubleshoot weak or nonspecific signals when using FOLR2 antibody, FITC conjugated?

When encountering weak or nonspecific signals with FOLR2-FITC antibodies, consider the following troubleshooting approaches:

For Western blot applications specifically, FOLR2 appears as a band at approximately 38 kDa, and optimal results have been achieved using 1 μg/mL of antibody on PVDF membranes under reducing conditions .

What are the considerations for optimizing FITC conjugation to FOLR2 antibodies?

For researchers performing their own FITC conjugation to FOLR2 antibodies, several critical factors must be considered:

• Antibody Concentration: Maintain consistent antibody concentration (ideally ≥2 mg/mL) during conjugation, as this affects conjugation efficiency .

• FITC:Antibody Ratio: The optimal ratio typically yields 3-6 FITC molecules per antibody. Higher ratios can cause:

  • Solubility problems

  • Internal quenching (reduced brightness)

  • Potential conformational changes affecting antibody binding

• Conjugation Protocol Optimization:

  • Perform parallel conjugations with different FITC:antibody ratios

  • Evaluate each conjugate for:

    • Brightness (mean fluorescence intensity)

    • Signal-to-noise ratio

    • Binding specificity compared to unconjugated antibody

• Post-Conjugation Processing:

  • FITC is unstable once solubilized and should be used immediately for conjugation

  • Purification by size-exclusion chromatography is essential to remove unconjugated FITC

  • Measure the F/P (fluorophore to protein) ratio by spectrophotometry at 280 nm (protein) and 495 nm (FITC)

• Storage Conditions:

  • Store in PBS at 4°C in the dark

  • Consider adding stabilizers (e.g., 1% BSA) for prolonged stability

  • Avoid repeated freeze-thaw cycles

How does FOLR2 antibody, FITC conjugated compare with other folate receptor targeting methods?

When comparing FOLR2-FITC antibodies with alternative folate receptor targeting approaches:

FOLR2-FITC antibodies offer the advantage of isoform specificity, distinguishing FOLR2 from the related FOLR1 and FOLR3 proteins despite their sequence homology of 68% and 79%, respectively . For functional studies examining folate uptake and metabolism, folate-conjugated fluorophores may provide more physiologically relevant data, while FOLR2-FITC antibodies excel at precise cellular identification and quantification of receptor expression levels.

What are the relative benefits of different immunofluorescence techniques when using FOLR2 antibody, FITC conjugated?

Different immunofluorescence techniques offer distinct advantages when working with FOLR2-FITC conjugated antibodies:

• Confocal Microscopy:

  • Provides high-resolution subcellular localization of FOLR2

  • Enables co-localization studies with other cellular markers

  • Reduces out-of-focus fluorescence

  • Particularly useful for visualizing FOLR2 distribution on cell surfaces and in cytoplasm

• Epifluorescence Microscopy:

  • Simpler setup and higher throughput

  • Better for initial screening and quantification

  • Less photobleaching than confocal

  • Suitable for routine FOLR2 detection in cultured cells

• Flow Cytometry:

  • Quantitative assessment of FOLR2 expression levels

  • High-throughput analysis of large cell populations

  • Allows for multiparameter analysis with other cellular markers

  • Enables sorting of FOLR2-positive cells for downstream applications

• Super-Resolution Microscopy:

  • Nanoscale visualization of FOLR2 clustering and organization

  • Reveals receptor distribution patterns not visible with conventional microscopy

  • Can track single-molecule dynamics of FOLR2

  • Requires specialized equipment and optimization

For studying FOLR2 in neutrophils, validation studies have employed fixed-cell immunofluorescence with FOLR2-specific antibodies at 10 μg/mL for 3 hours at room temperature, counterstained with DAPI for nuclear visualization .

How is FOLR2 antibody, FITC conjugated being used in development of new genotype-phenotype linked antibody screening methods?

Recent innovations in antibody discovery leverage FITC-conjugated antibodies for direct linkage of antigen-antibody binding with gene encoding. While not specifically developed for FOLR2, these methodologies are applicable to FOLR2 antibody development and screening:

• Membrane-Bound Antibody Expression Systems:

  • New dual-expression vector systems enable rapid screening of recombinant monoclonal antibodies through in-vivo expression of membrane-bound antibodies

  • These systems link antigen-binding features directly to Ig DNA sequence information

  • FOLR2-FITC can be used as a probe to identify and select cells expressing FOLR2-binding antibodies

  • This approach facilitates isolation of high-affinity antibodies within 7 days, compared to traditional methods requiring weeks

• Advantages for FOLR2 Research:

  • Membrane Ig expression directly links antigen-binding properties to the Ig DNA sequence

  • Dual Ig expression vectors link heavy and light chain genes, reducing preparation time

  • Golden Gate Cloning technology generates reliable plasmid clones quickly

  • Fluorescence intensity profiles directly reflect antibody affinity

• Application to FOLR2-Specific Antibody Development:

  • Could accelerate discovery of FOLR2-targeting antibodies for therapeutic applications in cancer and inflammatory diseases

  • Enables rapid screening of cross-reactive antibodies that might target multiple folate receptor family members

  • Facilitates affinity maturation studies through directed evolution approaches

This emerging technology streamlines mAb isolation for both therapeutic and diagnostic applications, which is particularly relevant for developing highly specific FOLR2 antibodies .

What role does FOLR2 play in methotrexate transport, and how can FITC-conjugated antibodies help investigate this mechanism?

FOLR2 may play a significant role in the transport of methotrexate in synovial macrophages of rheumatoid arthritis patients, representing an important area for investigation . FITC-conjugated FOLR2 antibodies can be instrumental in elucidating this mechanism through:

• Receptor Occupancy Studies:

  • Competitive binding assays between methotrexate and FOLR2-FITC antibodies

  • Displacement curves to determine binding affinities

  • Flow cytometric analysis of receptor saturation at various methotrexate concentrations

• Transport Mechanism Investigation:

  • Time-course studies of FOLR2 internalization with and without methotrexate

  • Co-localization studies using FOLR2-FITC and labeled methotrexate

  • Analysis of transport kinetics through quantitative imaging

• Patient-Derived Sample Analysis:

  • Comparison of FOLR2 expression levels in responders vs. non-responders to methotrexate therapy

  • Correlation of FOLR2 density (measured by FITC-antibody binding) with clinical outcomes

  • Ex vivo methotrexate uptake studies in patient-derived macrophages

• Experimental Design Considerations:

  • Use parallel samples with FOLR1 and FOLR3 antibodies to assess isoform specificity

  • Include controls with folate-free media to maximize FOLR2 expression

  • Consider pH dependency of folate binding when designing transport studies

Understanding this mechanism could lead to improved therapeutic approaches for rheumatoid arthritis and potentially other inflammatory conditions where methotrexate is used as a treatment.