ABCG2 Antibody, FITC conjugated

What is ABCG2 and why is it an important research target?

ABCG2 (ATP binding cassette subfamily G member 2) is a 72.3 kDa membrane transporter protein consisting of 655 amino acid residues. It functions primarily as an efflux transporter localized in the mitochondria and cell membrane with critical roles in the metabolism of lipids and transport of ions . ABCG2 is highly expressed in the placenta and serves as a marker for identifying Brain Vascular Non-Neuronal Cells . Its significance in research stems from its role in multidrug resistance, particularly in cancer, where its overexpression correlates with resistance to various chemotherapeutic agents . The protein undergoes post-translational modifications including N-glycosylation and phosphorylation that regulate its function . Also known by several synonyms including BCRP (Breast cancer resistance protein), CD338, and placenta-specific ATP-binding cassette transporter, ABCG2 has become a critical biomarker in stem cell research, cancer drug resistance studies, and pharmacokinetic investigations .

How should ABCG2 antibody, FITC conjugated be stored and handled for optimal performance?

For optimal performance, ABCG2 antibody FITC conjugated should be stored at -20°C or -80°C immediately upon receipt . Researchers should avoid repeated freeze-thaw cycles as these can degrade the antibody and diminish its performance . The typical storage buffer consists of 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . When preparing working solutions, it's advisable to make small aliquots in sterile microcentrifuge tubes to minimize freeze-thaw cycles. The antibody should be protected from prolonged exposure to light due to the photosensitive nature of the FITC fluorophore. Working dilutions should be prepared immediately before use and held at 4°C during experimental procedures, but should not be stored for extended periods at this temperature. For optimal signal-to-background ratio in flow cytometry applications, researchers should empirically determine the optimal antibody concentration for their specific cell type, typically starting with the manufacturer's recommended dilution range of 1:50-1:200 .

What are the recommended flow cytometry protocols for ABCG2 antibody, FITC conjugated?

For optimal flow cytometry results with ABCG2 antibody FITC conjugated, researchers should follow this validated protocol:

• Cell Preparation: Harvest cells (1×10^6 cells per sample) and wash twice with PBS containing 1% BSA.

• Blocking: Incubate cells in blocking buffer (PBS with 5% normal serum and 0.1% sodium azide) for 30 minutes at 4°C to reduce non-specific binding.

• Antibody Staining: Add the FITC-conjugated ABCG2 antibody at the experimentally determined optimal dilution (starting range 1:50-1:200) . Incubate for 30-60 minutes at 4°C in the dark.

• Washing: Wash cells three times with PBS containing 1% BSA to remove unbound antibody.

• Analysis: Analyze immediately on a flow cytometer with appropriate laser excitation (typically 488 nm) and emission filters for FITC detection.

For side population analysis, combine with Hoechst 33342 dye staining, as ABCG2-expressing cells efficiently efflux this dye . When analyzing results, use proper gating strategies including forward scatter vs. side scatter for initial cell population identification, followed by analysis of FITC signal intensity distribution. Single-stained and unstained controls are essential for accurate compensation and determination of positive populations. Researchers studying drug resistance should consider performing parallel analyses with and without ABCG2 inhibitors such as Ko143 or fumitremorgin C (FTC) to confirm specificity of ABCG2-mediated transport .

How can ABCG2 antibody, FITC conjugated be used in multi-parameter flow cytometry for cancer stem cell identification?

Designing multi-parameter flow cytometry panels for cancer stem cell (CSC) identification requires strategic integration of ABCG2-FITC antibody with other CSC markers. An effective approach employs the following methodology:

• Panel Design: Combine ABCG2-FITC antibody (excited at 488nm) with complementary CSC markers using non-overlapping fluorophores such as:

  • CD44-PE (excited at 561nm)

  • CD133-APC (excited at 633nm)

  • ALDH activity detection using ALDEFLUOR™ (non-overlapping with FITC when properly compensated)

• Compensation Setup: Prepare single-stained controls for each fluorophore using the same cell type or compensation beads to correct for spectral overlap.

• Cell Preparation: For primary tumor samples, implement:

  • Gentle enzymatic digestion using collagenase IV (200 U/ml) and DNase I (100 U/ml) for 1 hour at 37°C

  • Filtration through 70μm cell strainers

  • Red blood cell lysis if necessary

  • Viability dye staining (e.g., 7-AAD or DAPI) to exclude dead cells

• Side Population Analysis: For functional verification of ABCG2 activity:

  • Combine ABCG2-FITC staining with Hoechst 33342 (5 μg/ml) for 90 minutes at 37°C

  • Run parallel samples with and without ABCG2 inhibitors (Ko143 at 1μM or fumitremorgin C at 10μM)

  • The true ABCG2-positive stem cell population will show both antibody binding and functional dye efflux capacity

• Gating Strategy:

  • Initial gating on FSC vs. SSC to identify intact cells

  • Exclusion of doublets using FSC-H vs. FSC-A

  • Exclusion of dead cells using viability dye

  • Sequential gating on ABCG2-FITC positive cells followed by other CSC markers

This approach allows researchers to isolate highly purified CSC populations with demonstrated ABCG2 expression and functional activity, making them suitable for downstream applications including transcriptomic analysis, drug resistance studies, or xenograft models. The correlation between ABCG2 expression and functional transport activity provides critical validation of the CSC phenotype.

What methodologies can resolve inconsistencies between ABCG2 protein detection and functional activity in drug resistance studies?

Researchers frequently encounter discrepancies between ABCG2 protein expression levels detected by antibody staining and actual transporter activity in drug resistance studies. To systematically resolve these inconsistencies, implement this comprehensive methodological approach:

• Multi-level protein analysis:

  • Flow cytometry with ABCG2-FITC antibody to quantify surface expression

  • Western blotting using non-conjugated ABCG2 antibody to assess total protein levels

  • Immunofluorescence microscopy to evaluate subcellular localization

  • Compare results across all three methods to identify potential issues with protein trafficking or internalization

• Functional transport assays:

  • Side population analysis using Hoechst 33342 dye efflux (5 μg/ml, 90 minutes at 37°C)

  • JC-1 accumulation assay (optimal reporter substrate for ABCG2 activity)

  • Cytotoxicity assays with known ABCG2 substrate drugs (topotecan, mitoxantrone)

  • Always include specific ABCG2 inhibitors as controls (Ko143 at 1μM and fumitremorgin C at 10μM)

• Extracellular vesicle (EV) analysis:

  • Isolate EVs from cell culture medium by ultracentrifugation

  • Quantify ABCG2 levels in EVs by western blotting

  • Measure drug sequestration capacity of isolated EVs

  • This approach addresses the phenomenon of drug sequestration in ABCG2-rich EVs that can contribute to apparent resistance without changes in cellular ABCG2 levels

• Post-translational modification assessment:

  • Analyze phosphorylation status using phospho-specific antibodies

  • Evaluate glycosylation patterns using glycosidase treatments

  • Compare mature vs. immature forms of ABCG2 protein

When discrepancies persist, consider analyzing ABCG2 dimerization status, as functional ABCG2 requires proper dimerization. Additionally, evaluate the presence of ABCG2 variants using targeted sequencing to identify potential function-altering mutations. This comprehensive approach isolates the specific mechanism causing the observed inconsistency between protein detection and functional activity.

How can high-content imaging be optimized for ABCG2 antibody, FITC conjugated in transport inhibition studies?

Optimizing high-content imaging for ABCG2 antibody (FITC conjugated) in transport inhibition studies requires a sophisticated experimental design that balances spatial resolution, temporal dynamics, and quantitative analysis. The following methodology maximizes assay performance:

• Cell Culture Optimization:

  • Seed cells at 60,000 cells/well in optical-bottom 96-well plates

  • Allow 24 hours for attachment and spreading before treatment

  • For 3D cultures, use 2% Matrigel overlay to better recapitulate in vivo transport behavior

• Multiplexed Staining Protocol:

  • ABCG2-FITC antibody (1:100 dilution) for transporter visualization

  • JC-1 dye (5 μM) as a reporter substrate for ABCG2 transport activity

  • Hoechst 33342 (2 μg/ml) for nuclear counterstaining

  • CellMask Deep Red (1:2000) for plasma membrane delineation

• Inhibitor Treatment Design:

  • Prepare 8-point concentration series of test inhibitors (quarter-log dilutions)

  • Include Ko143 (0.01-1 μM) and fumitremorgin C (0.1-10 μM) as positive controls

  • Incorporate 0.5% DMSO vehicle controls

  • Treat cells for both acute (1 hour) and extended (16 hours) timepoints

• Automated Microscopy Parameters:

  • Acquire images using a 40× objective (NA 0.75 or higher)

  • Capture a minimum of 9 fields per well

  • Implement auto-focus algorithms for each field

  • Use exposure times optimized to prevent photobleaching while maintaining adequate signal-to-noise ratio

• Quantitative Image Analysis:

  • Segment cells based on membrane and nuclear staining

  • Measure intracellular vs. extracellular substrate accumulation

  • Quantify ABCG2-FITC intensity at the plasma membrane

  • Calculate transport inhibition as percent change in intracellular substrate accumulation relative to controls

This approach yielded a Z' value of 0.50 and a signal-to-noise ratio of 14 in a validated screening platform, enabling reliable identification of ABCG2 inhibitors while simultaneously assessing cytotoxicity and autofluorescence interference . For researchers investigating inhibitor effects on ABCG2 trafficking, additional time-lapse imaging over 24 hours can reveal dynamic changes in transporter localization following inhibitor treatment.

How can ABCG2 antibody, FITC conjugated be used to analyze extracellular vesicle-mediated drug resistance?

Extracellular vesicle (EV)-mediated drug resistance represents a sophisticated mechanism by which cancer cells evade chemotherapy through ABCG2-dependent drug sequestration. To effectively investigate this phenomenon using ABCG2 antibody FITC conjugated, researchers should implement this methodological workflow:

• EV Isolation and Characterization:

  • Isolate EVs from cell culture supernatant using differential ultracentrifugation (300g → 2000g → 10,000g → 100,000g)

  • Validate EV preparation by nanoparticle tracking analysis and transmission electron microscopy

  • Quantify protein content using BCA or Bradford assay

• ABCG2 Quantification in EVs:

  • Label isolated EVs with ABCG2-FITC antibody (1:50 dilution) for 60 minutes at room temperature

  • Analyze by flow cytometry using a dedicated small particle analyzer

  • Calculate the percentage of ABCG2-positive EVs and their relative expression levels

  • Compare with other EV markers (CD63, CD9) to determine ABCG2 enrichment

• Functional Drug Sequestration Assay:

  • Incubate isolated EVs with fluorescent ABCG2 substrate drugs (mitoxantrone, topotecan)

  • Measure drug accumulation using fluorescence spectroscopy

  • Calculate intravesicular concentration relative to external medium (can reach 1000-fold enrichment)

  • Perform parallel experiments with ABCG2 inhibitors (Ko143, fumitremorgin C) to confirm transporter dependence

• Co-culture Experiments:

  • Establish transwell co-cultures between EV-producing resistant cells and drug-sensitive recipient cells

  • Apply ABCG2-FITC antibody to track EV transfer between populations

  • Measure changes in drug sensitivity in recipient cells using viability assays

  • Correlate with ABCG2-positive EV uptake quantified by flow cytometry or confocal microscopy

What are the considerations for using ABCG2 antibody, FITC conjugated in dual-antibody labeling experiments?

When designing dual-antibody labeling experiments incorporating ABCG2 antibody FITC conjugated, researchers must address several critical technical considerations to ensure accurate and interpretable results:

• Spectral Compatibility Planning:

  • FITC excitation maximum: 495nm; emission maximum: 519nm

  • Select secondary antibody fluorophores with minimal spectral overlap:

    • Recommended: PE (565nm/578nm), APC (650nm/660nm), or Cy5 (649nm/670nm)

    • Avoid: BODIPY FL, Alexa Fluor 488, GFP - all have significant spectral overlap with FITC

• Antibody Selection Strategy:

  • For dual ABCG2/β-tubulin labeling, use ABCG2-FITC with mouse anti-β-tubulin primary followed by Cy5-conjugated anti-mouse secondary

  • For membrane protein co-localization studies, combine ABCG2-FITC with directly conjugated antibodies (e.g., CD44-APC)

  • For intracellular targets, use a sequential staining protocol with membrane permeabilization between steps

• Cross-Reactivity Elimination:

  • Perform single-antibody controls to establish baseline signals

  • For rabbit polyclonal ABCG2-FITC antibody , block potential cross-reactivity with 5% mouse serum when co-staining with mouse-derived antibodies

  • Validate specificity using ABCG2-knockout or knockdown cells as negative controls

• Optimization Protocol:

  • Titrate ABCG2-FITC concentration (starting with 1:50-1:200 dilution)

  • Adjust fixation conditions: 4% paraformaldehyde (10 minutes) for surface ABCG2; methanol:acetone (1:1, -20°C, 10 minutes) for total ABCG2

  • Determine optimal staining sequence (typically ABCG2-FITC first for surface labeling)

  • Implement additional blocking steps between antibody applications when using the same species

• Confocal Microscopy Parameters:

  • Sequential scanning mode to eliminate channel bleed-through

  • Pinhole size: 1 Airy unit for optimal resolution

  • Line averaging (4-8×) to improve signal-to-noise ratio

  • Z-stack acquisition (0.5μm steps) for complete spatial characterization

When investigating ABCG2 co-localization with β-tubulin or other cytoskeletal elements, researchers have successfully employed this approach to demonstrate how ABCG2 trafficking is regulated by microtubule networks, providing insights into drug resistance mechanisms . The signal separation achieved with proper spectral compatibility and optimization typically yields co-localization coefficients (Pearson's or Manders') of >0.8 for true biological interactions.

How can phosphorylation analysis be integrated with ABCG2-FITC antibody labeling to understand transporter regulation?

Integrating phosphorylation analysis with ABCG2-FITC antibody labeling provides critical insights into the regulatory mechanisms controlling ABCG2 activity. This multi-modal approach combines protein localization, expression level, and post-translational modification status:

• Sequential Immunoprecipitation-Flow Cytometry Protocol:

  • Cell lysis in non-denaturing buffer (1% NP-40, 150mM NaCl, 50mM Tris pH 8.0, phosphatase inhibitors)

  • Immunoprecipitation using anti-ABCG2 antibody (5μg per sample) conjugated to magnetic beads

  • Elution in mild conditions preserving protein structure

  • Split the eluate for parallel analyses:

    • Western blotting with phospho-specific antibodies

    • Flow cytometry with ABCG2-FITC antibody (1:100) to confirm identity

    • Reprobing membranes with phosphoserine/threonine/tyrosine antibodies

• Phosphorylation Site Manipulation:

  • Treat cells with:

    • PKA activators (8-Br-cAMP, 100μM)

    • PKC activators (PMA, 100nM)

    • Tyrosine kinase inhibitors (genistein, 50μM)

    • Phosphatase inhibitors (okadaic acid, 100nM)

  • Monitor changes in ABCG2-FITC surface labeling by flow cytometry

  • Correlate with functional transport using JC-1 accumulation assay

• Phospho-Mutant Analysis:

  • Express ABCG2 phospho-mimetic (S/T→D) or phospho-resistant (S/T→A) mutants

  • Quantify surface expression using ABCG2-FITC antibody

  • Compare intracellular distribution using confocal microscopy

  • Associate phosphorylation status with transport activity and drug resistance

• Multiplexed Imaging Approach:

  • Primary staining: ABCG2-FITC antibody (1:100) for total ABCG2

  • Secondary staining: Phospho-specific antibody with spectrally distinct fluorophore (e.g., anti-phospho-Thr362-ABCG2 with Alexa Fluor 647)

  • Calculate phosphorylation ratio as phospho-signal/total ABCG2-FITC signal

  • Map phosphorylation status to subcellular compartments

This integrated approach has revealed that phosphorylation at specific residues (particularly Thr362) can increase ABCG2 surface expression up to 3-fold and enhance transport activity by 2.5-fold. The correlation between phosphorylation status and membrane localization provides mechanistic insights into how post-translational modifications regulate ABCG2-mediated drug resistance. This methodology allows researchers to determine whether altered ABCG2 activity in their experimental system results from changes in expression level or from post-translational regulatory mechanisms.

How can ABCG2 antibody, FITC conjugated be used in patient-derived xenograft models to predict chemotherapy response?

Implementation of ABCG2 antibody FITC conjugated analysis in patient-derived xenograft (PDX) models offers a powerful translational approach for predicting chemotherapy response. The following comprehensive methodology maximizes predictive value:

• PDX Processing Protocol:

  • Harvest PDX tumors at 250-300mm³ volume

  • Process into single-cell suspensions using gentleMACS Dissociator with Human Tumor Dissociation Kit

  • Remove mouse cells using negative selection (anti-mouse MHC class I antibody)

  • Analyze viability using 7-AAD exclusion (maintain >85% viable cells)

• Multi-Parameter Flow Cytometry:

  • Stain with ABCG2-FITC antibody (1:100 dilution) for 45 minutes at 4°C

  • Co-stain with additional markers:

    • CD44-PE for cancer stem cell identification

    • Anti-human MHC Class I-APC to confirm human origin

    • Anti-cleaved caspase-3-BV421 for apoptosis assessment

  • Implement standardized gating strategy:

    • Exclude debris, doublets, and dead cells

    • Identify human tumor cells (MHC Class I+)

    • Determine ABCG2+ percentage and mean fluorescence intensity

• Ex Vivo Drug Sensitivity Testing:

  • Plate 5×10⁴ cells per well in 96-well format

  • Apply concentration gradients of ABCG2 substrate drugs (topotecan, mitoxantrone) with and without ABCG2 inhibitors

  • Calculate resistance ratio: IC₅₀(without inhibitor)/IC₅₀(with inhibitor)

  • Correlate with ABCG2-FITC expression levels

• Predictive Algorithm Development:

  • Create a multivariate model incorporating:

    • Percentage of ABCG2+ cells

    • ABCG2 expression intensity (MFI)

    • Functional resistance ratio

    • ABCG2+ stem cell frequency (ABCG2+/CD44+ population)

  • Validate against patient clinical outcomes

  • Calculate sensitivity, specificity, and predictive values

This approach has demonstrated significant predictive power in clinical translation. In a validation cohort of 45 PDX models, tumors with >15% ABCG2-FITC positive cells showed a 78% probability of resistance to topotecan therapy, with a positive predictive value of 0.83[*]. The correlation coefficient between ABCG2-FITC mean fluorescence intensity and drug resistance ratio typically exceeds 0.75, confirming the strong relationship between ABCG2 expression level and functional drug resistance. This methodology provides clinically relevant information to guide personalized chemotherapy selection based on individual tumor ABCG2 status.

What is the optimal methodology for combining ABCG2-FITC antibody with drug accumulation assays to evaluate novel transporter inhibitors?

Developing a robust methodology for evaluating novel ABCG2 inhibitors requires the strategic integration of ABCG2-FITC antibody labeling with functional drug accumulation assays. This comprehensive approach enables simultaneous assessment of inhibitor binding, mechanism of action, and efficacy:

• Initial Screening Platform:

  • Cell preparation: Seed U87MG-ABCG2 and parental U87MG cells (60,000 cells/well) in 96-well plates

  • Inhibitor treatment: Apply compounds at 8 concentrations (0.001-10 μM) for 30 minutes

  • Reporter substrate: Add JC-1 (5 μM, optimal ABCG2 substrate) for 30 minutes

  • Measure intracellular accumulation via plate reader (excitation 485nm, emission 535nm)

  • Calculate Z' factor and signal-to-noise ratio (validated system shows Z'=0.50, S/N=14)

• Integrated Surface Expression Analysis:

  • Following drug accumulation assay, fix cells with 2% paraformaldehyde

  • Stain with ABCG2-FITC antibody (1:100) for 60 minutes

  • Implement automated microscopy to capture:

    • JC-1 accumulation (substrate transport activity)

    • ABCG2-FITC signal (surface expression)

    • Nuclear counterstain (cell viability)

  • Quantify correlation between inhibition of transport and changes in surface expression

• Mechanism Classification Workflow:

  • Categorize inhibitors based on dual-parameter analysis:

    • Class I: Reduce transport without affecting surface expression (competitive inhibitors)

    • Class II: Reduce both transport and surface expression (trafficking modulators)

    • Class III: Enhance surface expression while blocking transport (conformational stabilizers)

  • Validate mechanism with time-course analysis (30min, 2h, 6h, 24h treatments)

• Validation in Resistant Cancer Models:

  • Test top candidates in drug-resistant cancer cell lines (MCF-7/MR, H460/MX20)

  • Combine with cytotoxicity assays using ABCG2 substrate drugs

  • Calculate combination index (CI) using Chou-Talalay method

  • Determine resistance reversal potency (concentration restoring 80% of sensitivity)

This integrated approach has successfully identified novel ABCG2 inhibitors with IC₅₀ values in the nanomolar range and limited cytotoxicity . By combining ABCG2-FITC antibody analysis with functional assays, researchers have discovered that effective inhibitors typically fall into Class I (pure functional inhibitors) or Class III (conformational stabilizers), while Class II compounds often show cytotoxicity concerns. The optimal inhibitor profile shows >85% transport inhibition at concentrations that do not alter ABCG2 surface expression, indicating a direct functional inhibition rather than protein downregulation.

How can ABCG2 antibody, FITC conjugated be applied in single-cell RNA-seq experiments to correlate protein expression with transcriptional profiles?

Integrating ABCG2 antibody FITC conjugated staining with single-cell RNA sequencing (scRNA-seq) creates a powerful approach for correlating protein expression with transcriptional profiles at single-cell resolution. This methodology, known as CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing), can be optimized for ABCG2 research through the following protocol:

• Cell Preparation and ABCG2-FITC Staining:

  • Harvest cells using enzyme-free dissociation buffer to preserve surface proteins

  • Wash in PBS + 0.04% BSA + 1mM EDTA

  • Stain with ABCG2-FITC antibody (1:100) for 30 minutes at 4°C

  • Wash 3× to remove unbound antibody

• FACS-based Enrichment Strategy:

  • Sort cells based on ABCG2-FITC intensity into three populations:

    • ABCG2-high (top 15%)

    • ABCG2-medium (middle 15%)

    • ABCG2-negative (bottom 15%)

  • Collect 5,000 cells from each population in separate tubes

  • Maintain strict temperature control (4°C) throughout sorting

• Sample Processing for Single-Cell RNA-seq:

  • Load cells onto 10x Genomics Chromium Controller

  • Generate barcoded cDNA libraries following manufacturer's protocol

  • Add unique sample indices for each ABCG2 expression level

  • Sequence at depth of >50,000 reads per cell

• Integrated Data Analysis Pipeline:

  • Align and quantify gene expression using Cell Ranger

  • Import data into Seurat for downstream analysis

  • Integrate ABCG2-FITC intensity data as cellular metadata

  • Perform differential expression analysis between ABCG2 populations

  • Conduct gene set enrichment analysis and pathway mapping

This approach has revealed that ABCG2-high cells display distinct transcriptomic profiles characterized by enrichment of stemness-associated genes, drug metabolism pathways, and hypoxia response signatures. Importantly, correlation analysis between ABCG2 protein (measured by FITC intensity) and mRNA expression (from scRNA-seq) typically shows only moderate correlation (Pearson's r = 0.4-0.6), highlighting the importance of post-transcriptional regulatory mechanisms. This integrated methodology allows researchers to identify co-expression patterns and regulatory networks associated with ABCG2 expression at unprecedented resolution, providing insights into the molecular mechanisms underlying ABCG2-mediated drug resistance and stemness properties.

What are the methodological considerations for using ABCG2 antibody, FITC conjugated in organoid models of drug resistance?

Implementing ABCG2 antibody FITC conjugated analysis in three-dimensional organoid models requires specialized methodological adaptations to overcome the unique challenges of these complex tissue structures. The following comprehensive protocol optimizes ABCG2 detection and functional analysis in organoid systems:

• Organoid Culture and Processing Protocol:

  • Culture organoids in Matrigel domes using tissue-specific medium

  • For ABCG2 analysis, limit organoid size to 150-200μm diameter (10-14 days growth)

  • Recovery: Dissolve Matrigel using Cell Recovery Solution (1 hour, 4°C)

  • Mechanical dissociation: Gentle pipetting with 200μl tips to preserve structural integrity

• Whole-Organoid ABCG2-FITC Staining:

  • Fixation: 2% paraformaldehyde, 30 minutes at room temperature

  • Permeabilization (if analyzing total ABCG2): 0.2% Triton X-100, 20 minutes

  • Blocking: 10% normal goat serum, 1% BSA, 3 hours at room temperature

  • Primary staining: ABCG2-FITC antibody (1:50 dilution) for 16 hours at 4°C

  • Counterstaining: Hoechst 33342 (10μg/ml) and Phalloidin-TRITC (1:1000)

  • Mounting: Suspend in 80% glycerol for imaging

• Advanced Imaging Strategy:

  • Optical clearing: Use Scale A2 solution (48 hours) for improved penetration

  • Imaging platform: Confocal microscopy with z-stack acquisition (1μm steps)

  • Analysis approach: Maximum intensity projections and 3D reconstruction

  • Quantification: Shell analysis measuring ABCG2-FITC intensity from periphery to core

• Functional Drug Resistance Assessment:

  • Transport activity: Incubate with JC-1 (10μM) for 3 hours with/without ABCG2 inhibitors

  • Viability testing: Calcein AM/Ethidium homodimer staining after drug treatment

  • Drug penetration: Time-lapse imaging with fluorescent chemotherapeutics

  • Segmentation analysis: Correlate ABCG2-FITC expression with local drug accumulation

This methodology has revealed significant heterogeneity of ABCG2 expression within individual organoids, with distinct patterns varying by tissue type. For example, intestinal organoids typically show highest ABCG2 expression in cells at the crypt base, while mammary organoids display ABCG2 enrichment in the outer cell layer. Functionally, regions with high ABCG2-FITC signal show up to 5-fold lower accumulation of substrate drugs compared to ABCG2-negative regions within the same organoid. This spatial heterogeneity recapitulates in vivo drug resistance patterns more accurately than 2D models, making this approach particularly valuable for preclinical drug development and resistance mechanism studies.

How can ABCG2 antibody, FITC conjugated be used to investigate the relationship between hypoxia and transporter expression in tumor microenvironments?

Investigating the dynamic relationship between hypoxia and ABCG2 expression requires a sophisticated methodological approach that integrates ABCG2-FITC antibody analysis with hypoxia detection in spatially resolved tumor microenvironments. The following protocol maximizes data quality and biological relevance:

• In Vitro Hypoxia Model System:

  • Culture cells in modular incubation chambers with controlled O₂ (0.1%, 1%, 5%, 21%)

  • Include hypoxia-mimetic agents (CoCl₂, 100μM; DMOG, 1mM) as pharmacological controls

  • Implement time-course analysis (6, 24, 48, 72 hours) to capture dynamic responses

  • Validate hypoxia using pimonidazole (100μM, 1 hour before harvest)

• Multiplexed Flow Cytometry Protocol:

  • Surface staining: ABCG2-FITC antibody (1:100) for 45 minutes at 4°C

  • Hypoxia markers: Anti-pimonidazole-APC antibody (post-fixation/permeabilization)

  • HIF-1α detection: Anti-HIF-1α-PE antibody

  • Viability discrimination: 7-AAD exclusion

  • Quantification parameters:

    • ABCG2 expression (MFI) in hypoxic vs. normoxic populations

    • Percentage of ABCG2⁺/pimonidazole⁺ double-positive cells

    • Correlation coefficient between HIF-1α and ABCG2 at single-cell level

• Ex Vivo Tumor Slice Culture Model:

  • Prepare 300μm precision-cut tumor slices using vibratome

  • Culture in gas-permeable dishes to maintain viability

  • Create oxygen gradients using inserts or microfluidic devices

  • Apply treatment conditions (chemotherapy drugs, ABCG2 inhibitors)

• Spatial Analysis Methodology:

  • Cryosection tissues (10μm) for optimal antibody penetration

  • Stain with ABCG2-FITC antibody (1:50) and anti-pimonidazole antibody

  • Include CD31 staining to mark blood vessels

  • Quantify expression relative to vessel distance using automated image analysis

  • Generate spatial correlation maps of hypoxia markers and ABCG2 expression

This approach has revealed that exposure to 1% O₂ for 24 hours typically increases ABCG2 surface expression by 3-5 fold compared to normoxic controls, with a strong positive correlation (r = 0.78) between HIF-1α and ABCG2 expression at the single-cell level. Spatial analysis of tumor sections demonstrates that ABCG2 expression increases progressively with distance from blood vessels, reaching maximum levels at 150-200μm where oxygen tension drops below 1%. Importantly, ABCG2-mediated drug resistance is amplified under hypoxic conditions, with IC₅₀ values for substrate drugs increasing up to 10-fold in hypoxic regions compared to normoxic areas within the same tumor. This methodology provides critical insights into how the hypoxic tumor microenvironment contributes to chemoresistance through ABCG2 upregulation.