FTL3 Antibody, FITC conjugated

What is FLT3 and why is it a significant research target?

FLT3 (Fms-like tyrosine kinase 3, also known as CD135 or FLK2) is a cell-surface receptor tyrosine kinase that plays a crucial role in hematopoietic stem/progenitor cell development. It acts as a receptor for the cytokine FLT3LG and regulates differentiation, proliferation, and survival of hematopoietic progenitor cells and dendritic cells . FLT3 is a significant research target because mutations resulting in constitutive activation of this receptor are commonly found in acute myeloid leukemia (AML) and acute lymphoblastic leukemia . Particularly, internal tandem duplication (ITD) mutations of FLT3 are associated with poor prognosis in AML patients, making FLT3 detection and targeting critically important in both basic research and clinical settings .

What are the key applications for FITC-conjugated FLT3 antibodies in research?

FITC-conjugated FLT3 antibodies are primarily used in flow cytometry (FC) and immunofluorescence (IF) applications . In flow cytometry, these antibodies enable researchers to quantify FLT3 expression on cell surfaces, identify specific cell populations expressing FLT3, and monitor changes in FLT3 expression under different experimental conditions. For immunofluorescence, FITC-conjugated FLT3 antibodies allow visualization of FLT3 localization within cells or tissues. The FITC fluorophore has excitation/emission maxima wavelengths of 495 nm/524 nm, making it compatible with standard fluorescence detection systems . These applications provide researchers with valuable tools for studying normal hematopoiesis, leukemic transformation, and potential therapeutic targets.

How should FITC-conjugated FLT3 antibodies be stored and handled to maintain optimal activity?

For optimal performance and stability, FITC-conjugated FLT3 antibodies should be stored at -20°C in appropriate buffer conditions . The commercial preparation typically includes a storage buffer containing 500 mM NaCl, 10 mM HEPES pH 7.0, 5 mM EDTA, and 0.09% sodium azide . When working with these antibodies:

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

• Protect from prolonged exposure to light as FITC is photosensitive

• Maintain cold chain during handling (use ice or refrigeration)

• Use appropriate dilution buffer for experiments (typically PBS with 1-5% BSA)

• Consider adding sodium azide (0.02-0.05%) to working solutions to prevent microbial contamination during longer storage

Properly maintained antibodies will retain specific binding activity and fluorescent properties, ensuring reliable experimental results.

How can FITC-conjugated FLT3 antibodies be optimized for detection of FLT3-ITD mutations in AML research?

Optimizing FITC-conjugated FLT3 antibodies for detection of FLT3-ITD mutations requires careful experimental design. While the antibody binds to the extracellular domain of FLT3 regardless of mutation status, researchers can leverage differential expression patterns and activation states:

• Titration experiments are essential to determine optimal antibody concentration that distinguishes between wild-type and ITD-mutated FLT3 expression levels

• Co-staining with phospho-specific antibodies targeting downstream signaling molecules can help identify constitutively active FLT3-ITD

• Develop a gating strategy that accounts for the typically higher surface expression of FLT3-ITD compared to wild-type FLT3

For quantitative analysis, establish a standardized mean fluorescence intensity (MFI) ratio between patient samples and control cells. Research indicates that FLT3-ITD positive cells often show 1.5-3 fold higher surface expression compared to wild-type counterparts . Additionally, time-course experiments following FLT3 inhibitor treatment can reveal differences in receptor internalization and degradation between wild-type and ITD-mutated FLT3, providing another distinguishing characteristic.

What are the most effective multiparameter flow cytometry panels incorporating FITC-conjugated FLT3 antibodies?

Effective multiparameter flow cytometry panels incorporating FITC-conjugated FLT3 antibodies should be designed based on research questions and cell populations of interest. For hematopoietic stem/progenitor cell analysis:

For leukemia research, panels should include:

• Markers for blast identification (CD34, CD117)

• Myeloid markers (CD33, CD13)

• Markers for minimal residual disease assessment

• Viability dye to exclude dead cells

When designing these panels, spectral overlap must be carefully considered, particularly between FITC and PE channels. Proper compensation controls are critical, and single-stained controls should be included in each experiment to account for day-to-day variations in instrument performance .

How can researchers combine FLT3 antibody detection with functional assays to assess FLT3 inhibitor efficacy?

Researchers can develop integrated protocols that combine FLT3 surface detection with functional readouts to comprehensively assess FLT3 inhibitor efficacy:

• Surface FLT3 quantification using FITC-conjugated antibodies to monitor receptor internalization following inhibitor treatment

• Intracellular phospho-flow cytometry to measure inhibition of downstream signaling molecules (pSTAT5, pERK, pAKT)

• Apoptosis assays (Annexin V/PI) to correlate FLT3 inhibition with cell death

• Cell cycle analysis to assess proliferation arrest

A methodological approach involves treating cells with titrated concentrations of FLT3 inhibitors (e.g., midostaurin, gilteritinib), followed by multiparameter analysis at defined time points (typically 1, 24, and 48 hours). This approach allows for correlation between surface FLT3 levels, signaling inhibition, and functional outcomes on a single-cell level.

The combination of 20D9-ADC (antibody-drug conjugate targeting FLT3) with TKI midostaurin has demonstrated strong synergistic effects in vitro and in vivo, leading to reduction of aggressive AML cells below detection limits . This synergy can be quantified using combination index calculations from flow cytometry data.

What are common technical challenges when using FITC-conjugated FLT3 antibodies and how can they be addressed?

Several technical challenges may arise when working with FITC-conjugated FLT3 antibodies:

• Low signal intensity: FITC has moderate brightness and may photobleach

  • Solution: Use higher antibody concentration after titration optimization

  • Consider longer acquisition time with reduced laser power

  • Implement signal amplification methods if needed

• High background fluorescence:

  • Solution: Optimize blocking protocols (use 10% normal serum from the same species as secondary antibody)

  • Increase washing steps and duration

  • Use appropriate negative controls to establish baseline fluorescence

• Inconsistent staining:

  • Solution: Standardize sample preparation protocols

  • Ensure consistent antibody storage conditions

  • Prepare fresh working dilutions for each experiment

• Autofluorescence in the FITC channel:

  • Solution: Include unstained controls to determine autofluorescence levels

  • Consider alternative fluorophores if cell populations show high intrinsic fluorescence

  • Implement autofluorescence subtraction during analysis

• Antibody internalization during staining:

  • Solution: Perform staining at 4°C rather than 37°C

  • Reduce incubation time if internalization is problematic

  • Add sodium azide to staining buffer to inhibit energy-dependent internalization

Implementing quality control measures such as using consistent positive controls (e.g., cell lines with known FLT3 expression levels) can help track and address these issues systematically .

How should researchers interpret complex FLT3 expression patterns in heterogeneous samples?

Interpreting complex FLT3 expression patterns in heterogeneous samples requires sophisticated analytical approaches:

• Establish clear gating hierarchies:

  • Begin with viability markers to exclude dead cells

  • Gate on relevant cell populations using lineage markers before analyzing FLT3 expression

  • Use isotype controls to set positive/negative boundaries

• Quantify expression using appropriate metrics:

  • Report both percentage of FLT3-positive cells and median fluorescence intensity

  • Calculate FLT3 index (MFI × % positive) for more comprehensive analysis

  • Consider density plots rather than simple histograms for better visualization

• Address bimodal or complex distributions:

  • When populations show distinct FLT3 expression levels, report metrics for each subpopulation

  • Use clustering algorithms (viSNE, FlowSOM) for unbiased identification of cell populations

  • Consider trajectory analysis for developmental studies

• Account for contextual expression:

  • Analyze FLT3 expression in conjunction with differentiation markers

  • Compare expression patterns to established references

  • Track expression changes longitudinally when possible

In AML samples, for example, researchers should distinguish between leukemic blasts and residual normal progenitors, as both may express FLT3 but with different patterns and intensities . Sophisticated computational approaches can help deconvolute these complex patterns and identify clinically relevant subpopulations.

What controls are essential for validating FLT3 antibody specificity in research applications?

Rigorous validation of FLT3 antibody specificity requires multiple complementary controls:

• Positive controls:

  • Cell lines with confirmed FLT3 expression (e.g., MOLM-13, MV4-11)

  • Recombinant FLT3-expressing cells

  • Primary samples with known FLT3 status

• Negative controls:

  • FLT3-knockout or FLT3-negative cell lines

  • Isotype controls matched to antibody class and concentration

  • FLT3-blocking experiments using unlabeled antibodies or recombinant FLT3L

• Technical controls:

  • Single-stained compensation controls

  • Fluorescence-minus-one (FMO) controls

  • Titration series to determine optimal concentration

• Biological validation:

  • Correlation with genomic FLT3 status (wild-type vs. mutated)

  • Agreement with alternative detection methods (Western blot, qPCR)

  • Expected biological responses (e.g., receptor downregulation following ligand stimulation)

• Cross-reactivity assessment:

  • Testing antibody against related receptor tyrosine kinases

  • Species specificity confirmation if working with non-human models

Documenting these validation steps is essential for ensuring reproducible and reliable research outcomes. The antibody's specificity should be verified in the specific experimental context and cell types being studied .

How are FITC-conjugated FLT3 antibodies being integrated with single-cell technologies for leukemia research?

FITC-conjugated FLT3 antibodies are increasingly being integrated with cutting-edge single-cell technologies to advance leukemia research:

• Single-cell RNA sequencing (scRNA-seq) with protein detection:

  • Index sorting allows correlation of FLT3 surface expression with transcriptional profiles

  • CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) approaches incorporate FLT3 antibodies to simultaneously detect surface protein and gene expression

  • This integration reveals heterogeneity within FLT3-positive populations and identifies co-expression patterns with other markers

• Mass cytometry (CyTOF) applications:

  • While not using FITC directly, parallel workflows use metal-tagged FLT3 antibodies for high-dimensional phenotyping

  • These approaches allow simultaneous detection of >40 parameters, enabling comprehensive characterization of leukemic cells

  • FLT3 expression patterns can be correlated with signaling pathways and response to therapeutics

• Spatial transcriptomics and imaging:

  • FLT3 antibodies are being incorporated into spatial profiling techniques to understand microenvironmental interactions

  • Multiplexed imaging approaches combined with computational analysis reveal bone marrow niches supporting FLT3-mutated leukemic cells

These integrated approaches are particularly valuable for understanding heterogeneity in FLT3-mutated leukemias and identifying resistance mechanisms to FLT3 inhibitors. They also enable discovery of previously unrecognized cell states and developmental trajectories in normal and malignant hematopoiesis .

What are the latest developments in combining FLT3 antibody detection with therapeutic approaches?

Recent research has demonstrated several innovative approaches combining FLT3 antibody detection with therapeutic strategies:

• Antibody-drug conjugates (ADCs):

  • Novel FLT3-targeting ADCs such as 20D9-ADC have shown promising preclinical results

  • These conjugates demonstrated potent cytotoxicity against FLT3-expressing cells in vitro and significant tumor reduction in vivo

  • Combination of 20D9-ADC with the TKI midostaurin showed strong synergy, leading to reduction of aggressive AML cells below detection limits

• Monitoring therapeutic response:

  • FITC-conjugated FLT3 antibodies are being used to monitor changes in FLT3 expression during treatment

  • Flow cytometric assays combining FLT3 detection with apoptosis markers provide early indicators of response to FLT3 inhibitors

  • Post-transplant maintenance (PTM) using FLT3 inhibitors significantly reduces relapse risk and improves long-term outcomes in patients with FLT3-ITD mutations

• CAR-T and immunotherapy approaches:

  • FLT3 antibody fragments are being explored for chimeric antigen receptor (CAR) development

  • FITC-conjugated antibodies help identify optimal binding epitopes for immunotherapy development

  • Dual-targeting approaches combining FLT3 with other leukemia-associated antigens show promise for reducing escape mechanisms

• Minimal residual disease (MRD) detection:

  • High-sensitivity flow cytometry incorporating FLT3 antibodies enables detection of residual leukemic cells

  • Standardized protocols using FITC-conjugated FLT3 antibodies are being developed for MRD monitoring

  • Integration with other methodologies (e.g., molecular testing) improves sensitivity and specificity

These approaches highlight the evolution from purely diagnostic applications to integrated diagnostic-therapeutic strategies targeting FLT3 in leukemia .

How might advanced computational analysis enhance the value of FLT3 antibody-generated data?

Advanced computational analysis significantly enhances the value of data generated using FITC-conjugated FLT3 antibodies:

• Machine learning algorithms for pattern recognition:

  • Supervised learning approaches can identify subtle FLT3 expression patterns associated with therapeutic response

  • Unsupervised clustering algorithms reveal previously unrecognized cell populations with distinct FLT3 expression profiles

  • Deep learning models integrate multiple parameters to predict clinical outcomes based on FLT3 expression patterns

• Systems biology integration:

  • Network analysis incorporates FLT3 expression data with other molecular measurements to understand signaling dynamics

  • Pathway enrichment analyses contextualize FLT3 expression within broader biological processes

  • Multi-omics integration connects surface FLT3 levels with genomic, transcriptomic, and proteomic data

• Visualization tools for complex datasets:

  • Dimension reduction techniques (t-SNE, UMAP) enable visualization of high-dimensional flow cytometry data

  • Trajectory inference methods map developmental progressions based on FLT3 expression

  • Interactive visualization platforms allow researchers to explore complex relationships between FLT3 and other parameters

• Predictive modeling for therapeutic applications:

  • Computational models predict response to FLT3 inhibitors based on surface expression and mutation status

  • Pharmacodynamic models incorporate FLT3 antibody data to optimize dosing regimens

  • Virtual patient cohorts simulate treatment outcomes using FLT3 expression as a key parameter

These computational approaches transform descriptive FLT3 antibody data into actionable insights that guide research directions and therapeutic strategies. Implementation of standardized analysis pipelines further enhances reproducibility and facilitates meta-analyses across multiple studies .