SLC7A1 Antibody, FITC conjugated

What is SLC7A1 and what are its primary biological functions?

SLC7A1, also known as Cationic Amino Acid Transporter 1 (CAT1), functions as a high-affinity transporter for cationic amino acids in the Y+ system. It plays a crucial role in cellular amino acid homeostasis, particularly for arginine and phenylalanine. In ovarian cancer (OC) tissues, SLC7A1 is highly expressed and involved in amino acid metabolism essential for tumor development and progression . Research indicates that SLC7A1 participates in the transport of phenylalanine and arginine in epithelial ovarian cancer (EOC) cells, contributing to metabolic reprogramming that supports tumor growth . This transporter is not merely involved in nutrient acquisition but also appears to influence cellular processes including proliferation, migration, and drug resistance mechanisms. The protein's expression levels correlate with clinical outcomes in cancer patients, with higher expression generally associated with poorer survival outcomes, particularly in EOC .

What validation parameters should be assessed when first implementing SLC7A1 Antibody, FITC conjugated in a research protocol?

When implementing SLC7A1 Antibody, FITC conjugated in a new research protocol, comprehensive validation is essential to ensure reliable results. Researchers should first confirm antibody specificity through positive and negative controls, including tissues or cell lines with known high or low/absent SLC7A1 expression levels. Western blotting with the unconjugated version of the same antibody clone can help verify binding to the expected molecular weight protein (approximately 67-70 kDa for SLC7A1). For the FITC-conjugated antibody specifically, validation should include:

• Titration experiments to determine optimal antibody concentration

• Signal-to-noise ratio assessment across different fixation and permeabilization conditions

• Competitive binding assays using blocking peptides corresponding to the immunogen (AA 430-492)

• Comparison with alternative SLC7A1 antibody clones targeting different epitopes

• Confirmation of expected subcellular localization patterns

Since the antibody was produced using recombinant Human High affinity cationic amino acid transporter 1 protein (430-492AA) as immunogen , researchers should be particularly attentive to potential cross-reactivity with other proteins containing similar sequence motifs.

How can SLC7A1 Antibody, FITC conjugated be utilized to investigate the relationship between amino acid transport and cancer progression?

SLC7A1 Antibody, FITC conjugated can be employed in multiple experimental approaches to investigate amino acid transport and cancer progression. Flow cytometry can quantify SLC7A1 expression levels across different cancer cell populations and correlate expression with functional assays. Confocal microscopy using this antibody allows researchers to visualize SLC7A1 localization within cellular compartments and at the plasma membrane.

Research has demonstrated that SLC7A1 is involved in the transport of essential amino acids like phenylalanine and arginine in EOC cells . To investigate this relationship, researchers can employ the following methodology:

• Compare SLC7A1 expression levels (via FITC-conjugated antibody) with amino acid uptake measurements using an amino acid autoanalyzer

• Perform knockdown experiments of SLC7A1 and measure changes in specific amino acid concentrations

• Correlate SLC7A1 expression (measured by flow cytometry) with proliferation rates, migration capacity, and cisplatin resistance

For comprehensive analysis, researchers can utilize the Hitachi LA8080 amino acid automatic analyzer with dual-channel detection at wavelengths of 570 nm and 440 nm, as described in the literature . The 440 nm wavelength is particularly useful for detecting proline levels, while the complete amino acid profile can be assessed using both channels.

What approaches can resolve potential spectral overlap when using SLC7A1 Antibody, FITC conjugated in multi-parameter flow cytometry?

When using SLC7A1 Antibody, FITC conjugated in multi-parameter flow cytometry, spectral overlap presents a significant challenge that requires methodological solutions. To address this issue, researchers should implement the following strategies:

• Compensation matrix optimization: Prepare single-stained controls for each fluorophore in your panel alongside the FITC-conjugated SLC7A1 antibody. Use these to calculate the spectral spillover between channels and generate a comprehensive compensation matrix.

• Panel design considerations:

  • Avoid combining FITC with fluorophores having similar emission spectra (e.g., GFP, Alexa Fluor 488)

  • Assign FITC to antigens expressed at higher levels (like SLC7A1 in cancer cells) since FITC has moderate brightness

  • Place fluorophores with minimal spectral overlap on markers co-expressed with SLC7A1

• Alternative strategies:

  • Consider using spectral flow cytometry systems with unmixing algorithms

  • Employ fluorescence-minus-one (FMO) controls to establish accurate gating strategies

  • If persistent issues occur, inquire about the same antibody clone conjugated to alternative fluorophores with less spectral overlap

The polyclonal nature of the antibody targeting amino acids 430-492 should be considered when designing these experiments, as binding characteristics may differ slightly between antibody lots .

How does SLC7A1 expression correlate with immune cell infiltration in tumor microenvironments?

SLC7A1 expression demonstrates significant correlations with immune cell infiltration in tumor microenvironments, particularly in ovarian cancer. Research using the TIMER database has revealed that SLC7A1 overexpression is significantly positively correlated with levels of CD4+ memory resting cells, CD8+ effector memory cells, M0 macrophages, and cancer-associated fibroblasts (CAFs) in ovarian cancer (P < 0.05) . Conversely, SLC7A1 overexpression shows a significant negative correlation with CD4+ memory-activated cells (P < 0.05) .

To investigate these relationships, researchers can employ SLC7A1 Antibody, FITC conjugated in multi-parameter flow cytometry or immunofluorescence microscopy approaches:

• Flow cytometry protocol:

  • Prepare single-cell suspensions from tumor tissues

  • Use multi-color panels including SLC7A1-FITC alongside markers for:

    • T cell subsets (CD3, CD4, CD8, CD45RO, CD45RA)

    • Macrophage populations (CD68, CD163, CD206)

    • Cancer-associated fibroblasts (α-SMA, FAP, PDGFRβ)

  • Analyze correlations between SLC7A1 expression levels and immune cell percentages

• Immunofluorescence approaches:

  • Perform multiplex immunofluorescence on tumor sections

  • Quantify spatial relationships between SLC7A1+ cells and immune populations

  • Measure infiltration patterns and distances between cell populations

Cell immunofluorescence studies have indicated that SLC7A1 overexpression may affect the distribution of immune-infiltrating lymphocytes in tumors by inhibiting the expression of CCL4, providing a potential mechanism for SLC7A1's immunomodulatory effects .

What is the optimal sample preparation protocol for detecting SLC7A1 using FITC-conjugated antibodies in tumor tissue sections?

The optimal sample preparation protocol for detecting SLC7A1 using FITC-conjugated antibodies in tumor tissue sections involves several critical steps to ensure specific staining while preserving tissue architecture. Based on established protocols in the literature, the following methodology is recommended:

• Tissue collection and fixation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard histological procedures

  • Cut sections at 4-5 μm thickness onto positively charged slides

• Antigen retrieval:

  • Deparaffinize sections in xylene and rehydrate through graded alcohols

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Heat in a pressure cooker or microwave for 20 minutes, then cool to room temperature

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Apply protein block (e.g., 5% normal serum) for 30 minutes

  • Incubate with FITC-conjugated SLC7A1 antibody (targeting AA 430-492) at optimized dilution (typically 1:50-1:200) overnight at 4°C or 2 hours at room temperature

  • Wash three times with PBS

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI

  • Mount with anti-fade mounting medium designed for fluorescence preservation

Immunohistochemical scoring can be performed using a combined intensity and percentage scoring system as described in the literature, with staining intensity scores ranging from 0 (negative) to 3 (high) and percentage scores from 0 (no staining) to 3 (51%-100% staining) .

How can researchers quantitatively assess SLC7A1 expression changes in relation to cisplatin resistance?

Researchers can employ multiple quantitative approaches to assess SLC7A1 expression changes in relation to cisplatin resistance using FITC-conjugated antibodies. A comprehensive methodology includes:

• Flow cytometric quantification:

  • Culture cisplatin-sensitive and resistant cell lines

  • Stain cells with SLC7A1 Antibody, FITC conjugated

  • Measure mean fluorescence intensity (MFI) as a quantitative measure of SLC7A1 expression

  • Compare expression levels between sensitive and resistant populations

• Immunofluorescence microscopy with quantitative image analysis:

  • Perform immunofluorescence staining of sensitive and resistant cell lines or patient-derived samples

  • Capture standardized images using identical acquisition parameters

  • Utilize image analysis software to quantify:

    • Total SLC7A1-FITC signal intensity per cell

    • Subcellular localization patterns

    • Membrane-to-cytoplasm signal ratio

• Correlation with functional assays:

  • Measure cisplatin IC50 values across cell lines with varying SLC7A1 expression

  • Perform SLC7A1 knockdown experiments and assess changes in cisplatin sensitivity

  • Correlate SLC7A1 expression with apoptotic markers following cisplatin treatment

Research has shown that SLC7A1 knockdown reduces the resistance of cells to cisplatin, suggesting its potential role as a biomarker for predicting EOC progression and cisplatin resistance . The comprehensive quantitative assessment allows researchers to establish whether SLC7A1 expression can serve as a predictive biomarker for treatment response and potential therapeutic targeting.

What experimental approaches can link SLC7A1-mediated amino acid transport to cancer cell metabolism?

To establish connections between SLC7A1-mediated amino acid transport and cancer cell metabolism, researchers can implement several experimental approaches using SLC7A1 Antibody, FITC conjugated as a key tool:

• Amino acid uptake and metabolic profiling:

  • Sort SLC7A1-high and SLC7A1-low cell populations using FITC-conjugated antibodies and flow cytometry

  • Measure amino acid uptake rates using radiolabeled amino acids or metabolic tracers

  • Perform metabolomic analysis to identify differences in metabolic pathways between populations

• SLC7A1 manipulation and metabolic consequences:

  • Generate SLC7A1 knockdown and overexpression models

  • Quantify intracellular and extracellular amino acid concentrations using an amino acid autoanalyzer

  • Specifically monitor phenylalanine and arginine levels, as these have been identified as key SLC7A1 substrates in cancer cells

• Functional metabolic assays:

  • Measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using Seahorse technology

  • Perform isotope-labeled amino acid tracing experiments to track metabolic fates

  • Assess changes in metabolic enzyme activities in response to SLC7A1 modulation

• Correlative multi-omics approach:

  • Integrate SLC7A1 expression data (using FITC-antibody quantification) with:

    • Transcriptomic data on metabolic enzymes

    • Proteomic data on metabolic pathways

    • Metabolomic profiles of amino acid-derived metabolites

The amino acid autoanalyzer methodology described in the literature (Hitachi LA8080 with dual-channel detection at 570 nm and 440 nm) provides a robust approach for quantifying the impact of SLC7A1 on cellular amino acid pools . These comprehensive approaches can elucidate how SLC7A1-mediated amino acid transport contributes to metabolic reprogramming in cancer cells.

How can researchers address weak or inconsistent FITC signal when using SLC7A1 Antibody, FITC conjugated?

When encountering weak or inconsistent FITC signal with SLC7A1 Antibody, FITC conjugated, researchers should implement a systematic troubleshooting approach:

• Antibody and sample handling optimization:

  • Verify antibody storage conditions (4°C, protected from light)

  • Prepare fresh dilutions for each experiment

  • Titrate antibody concentration to identify optimal working dilution

  • Minimize exposure to light during all protocol steps

• Fixation and permeabilization optimization:

  • Test multiple fixatives (paraformaldehyde, methanol, acetone)

  • Optimize fixation time and temperature

  • Evaluate different permeabilization agents (Triton X-100, saponin, digitonin)

  • For membrane proteins like SLC7A1, consider milder permeabilization or use live-cell staining protocols

• Antigen retrieval enhancement:

  • Compare different antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize buffer composition, pH, and incubation times

  • For tissue sections, ensure complete deparaffinization and adequate retrieval time

• Signal amplification strategies:

  • Consider anti-FITC secondary antibodies conjugated to brighter fluorophores

  • Implement tyramide signal amplification (TSA) systems

  • Use anti-fade mounting media with signal preservatives

• Equipment calibration:

  • Ensure proper microscope filter sets for FITC detection

  • Optimize detector gain and exposure settings

  • Regularly calibrate flow cytometers with appropriate standards

Since the polyclonal SLC7A1 antibody targets amino acids 430-492, expression levels may vary depending on protein conformation and accessibility of this specific epitope region . Researchers should consider testing antibodies targeting different epitopes if signal issues persist.

What controls should be included when using SLC7A1 Antibody, FITC conjugated for quantitative immunofluorescence analysis?

When performing quantitative immunofluorescence analysis with SLC7A1 Antibody, FITC conjugated, a comprehensive set of controls is essential to ensure data validity and reproducibility:

• Antibody specificity controls:

  • Positive control: Cell lines or tissues with confirmed high SLC7A1 expression

  • Negative control: SLC7A1-knockout or low-expressing samples

  • Peptide blocking control: Pre-incubation of antibody with immunizing peptide (AA 430-492) to confirm binding specificity

  • Isotype control: Rabbit polyclonal IgG-FITC to assess non-specific binding

• Technical controls:

  • Autofluorescence control: Unstained sample to establish background fluorescence

  • Secondary-only control: For protocols using anti-FITC enhancement

  • Fixation control: Samples processed identically except for primary antibody addition

• Quantification controls:

  • Fluorescence standards: Calibration beads with known FITC molecules/bead

  • Dynamic range control: Serial dilutions of positive control lysate

  • Replicate controls: Technical and biological replicates to assess variability

• Imaging controls:

  • Flat-field correction: Uniform fluorescent slide to correct for illumination non-uniformities

  • Exposure series: Multiple exposures to ensure linearity of signal detection

  • Cross-channel bleeding control: Single-color controls to assess spectral overlap

For optimal quantification, researchers should establish standardized acquisition parameters and include slide-to-slide normalization controls to enable comparison across multiple experiments and potentially across different research groups investigating SLC7A1 expression in similar contexts.

How can SLC7A1 Antibody, FITC conjugated be integrated into multiplexed imaging approaches to study tumor heterogeneity?

SLC7A1 Antibody, FITC conjugated can be strategically integrated into multiplexed imaging approaches to study tumor heterogeneity through several advanced methodologies:

• Cyclic immunofluorescence (CycIF) integration:

  • Incorporate SLC7A1-FITC antibody into initial staining rounds

  • Image and record FITC signal coordinates

  • Chemically strip antibodies or quench FITC signal

  • Repeat staining with additional markers (up to 30-40 markers on the same tissue section)

  • Computational alignment and overlay of all markers

• Multiplexed ion beam imaging (MIBI) or Imaging Mass Cytometry (IMC) adaptation:

  • Conjugate metal isotopes to the same SLC7A1 antibody clone (instead of FITC)

  • Combine with 40+ other metal-labeled antibodies

  • Analyze with time-of-flight mass spectrometry

  • Generate high-dimensional spatial data connecting SLC7A1 expression with complex cellular phenotypes

• Spatial transcriptomics correlation:

  • Perform SLC7A1-FITC immunofluorescence on serial sections

  • Map SLC7A1 protein expression patterns

  • Correlate with spatial transcriptomics data from adjacent sections

  • Integrate amino acid transporter gene expression with metabolic pathway genes

• Digital spatial profiling approaches:

  • Use SLC7A1-FITC antibody alongside UV-photocleavable DNA barcode-tagged antibodies

  • Select regions of interest based on SLC7A1 expression patterns

  • Quantify multiple proteins from specific microenvironments

  • Correlate SLC7A1 with immune infiltration markers

Research has shown that SLC7A1 expression correlates with specific immune cell populations, including CD4+ memory resting cells, CD8+ effector memory cells, and M0 macrophages . Multiplexed imaging can reveal spatial relationships between SLC7A1-expressing cells and these immune populations, providing insights into the mechanistic basis for SLC7A1's role in the tumor immune microenvironment.

What methodological approaches can assess the relationship between SLC7A1 expression and immune checkpoint molecules?

To investigate the relationship between SLC7A1 expression and immune checkpoint molecules, researchers can implement several methodological approaches using SLC7A1 Antibody, FITC conjugated:

• Multi-parameter flow cytometry protocol:

  • Prepare single-cell suspensions from tumor tissues

  • Design panels combining SLC7A1-FITC with antibodies against:

    • PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT

    • T cell markers (CD3, CD4, CD8)

    • Exhaustion markers (TOX, EOMES)

  • Analyze correlations between SLC7A1 expression and checkpoint molecule levels

  • Sort cell populations based on SLC7A1 expression for further functional studies

• Multiplex immunofluorescence on tissue sections:

  • Perform sequential staining or spectral unmixing approaches

  • Include SLC7A1-FITC alongside checkpoint molecules

  • Quantify co-expression and spatial relationships

  • Correlate with patient treatment response data

• Transcriptional and protein correlation studies:

  • Sort SLC7A1-high versus SLC7A1-low populations using FITC-conjugated antibody

  • Perform transcriptome analysis (RNA-seq or NanoString)

  • Identify correlations between SLC7A1 and immune checkpoint gene expression

  • Validate at protein level using western blot or ELISA

• Functional relationship investigation:

  • Manipulate SLC7A1 expression (knockdown/overexpression)

  • Assess changes in checkpoint molecule expression

  • Investigate amino acid availability effects on checkpoint expression

  • Evaluate T cell functionality in co-culture systems

This methodological framework builds on research showing SLC7A1's correlation with immune cell infiltration , extending this to examine potential mechanisms by which amino acid transport might regulate immune checkpoint expression and function in the tumor microenvironment.