SLC7A2 Antibody, FITC conjugated

What is SLC7A2 and what biological functions does it serve?

SLC7A2 encodes the cationic amino acid transporter 2 (CAT2) with high affinity for L-arginine, a semi-essential amino acid involved in various physiological processes. This transporter plays crucial roles in cell division, proliferation, wound healing, and particularly in immune functions . The protein functions primarily by facilitating the transport of arginine across cell membranes, which is essential for nitric oxide (NO) synthesis, protein nitrosylation, and the production of other metabolites including urea, polyamines, proline, glutamate, creatine, and agmatine .

Recent research has demonstrated that SLC7A2 serves as a significant regulator of both innate and adaptive immunity in macrophages. This connection has particular relevance to neuroinflammatory conditions, as seen in Huntington's disease models where abnormal SLC7A2 upregulation correlates with dysregulated inflammatory responses . Additionally, SLC7A2 has been identified as a critical factor in myogenic differentiation, with expression levels gradually increasing during the differentiation process of C2C12 myoblasts .

What are the recommended applications for the SLC7A2-FITC conjugated antibody?

The FITC-conjugated anti-SLC7A2 antibody is specifically designed for immunofluorescent applications, with flow cytometry (FACS) being the primary recommended application . This antibody recognizes an extracellular epitope and can therefore detect the protein in living cells, making it particularly valuable for monitoring surface expression without cell permeabilization procedures .

The antibody demonstrates reactivity across multiple species including human, mouse, and rat samples, making it versatile for comparative studies across model systems . While flow cytometry represents the primary application, the affinity-purified nature of this antibody may potentially allow for adaptation to other immunofluorescence-based techniques where surface protein detection is required.

How should researchers validate the specificity of this antibody?

Validation of antibody specificity for SLC7A2 should follow a multi-step approach:

• Positive and negative controls: Compare staining between cell lines known to express SLC7A2 (such as activated macrophages or differentiating myoblasts) versus those with minimal expression.

• Knockdown verification: Perform siRNA-mediated knockdown of SLC7A2 as demonstrated in functional studies , then confirm reduced antibody binding via flow cytometry.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide ((C)KTYFKMNYTGLAE, corresponding to amino acids 151-163 of rat SLC7A2) before staining to demonstrate binding specificity.

• Cross-validation: Compare results with alternative SLC7A2 detection methods such as qRT-PCR or Western blotting with non-FITC conjugated antibodies targeting different epitopes.

How can researchers optimize flow cytometry protocols for SLC7A2-FITC detection?

Optimization of flow cytometry protocols for SLC7A2-FITC detection requires careful consideration of several factors:

Buffer Composition: Since SLC7A2 functions as an arginine transporter, buffer components can impact protein conformation and antibody accessibility. Use physiological buffers without excessive free arginine that might compete for transporter binding sites.

Live Cell Considerations:

• Maintain cell viability throughout the procedure as the antibody targets an extracellular epitope and is designed for live cell applications .

• Use appropriate viability dyes compatible with FITC to exclude dead cells during analysis.

• Minimize sample processing time to prevent endocytosis of the antibody-bound receptor.

Signal Optimization:

• Titrate the antibody concentration (starting from the recommended 50 μL per sample) to determine optimal signal-to-noise ratio .

• When designing multicolor panels, account for FITC spectral overlap with other fluorophores such as PE.

• Include unstained and single-color controls for proper compensation.

Gating Strategy:

• Implement a hierarchical gating approach starting with FSC/SSC to identify the population of interest.

• Use viability markers to exclude dead cells before analyzing SLC7A2 expression.

• Consider time-course measurements if studying dynamic changes in surface expression.

What methodological approaches are recommended for SLC7A2 knockdown studies?

Based on the research presented in the search results, SLC7A2 knockdown studies have yielded significant insights into its functional roles. Recommended methodological approaches include:

siRNA Transfection Protocol:

• Design siRNA specifically targeting SLC7A2 coding regions.

• Transfect cells at 50-70% confluency using appropriate transfection reagents.

• Allow 48 hours post-transfection before performing functional assays to ensure sufficient protein depletion .

Validation of Knockdown Efficiency:

• Quantitative RT-PCR to verify mRNA reduction (studies show successful knockdown is detectable at this level) .

• Western blotting to confirm protein depletion .

• Flow cytometry using the SLC7A2-FITC antibody to measure surface expression changes.

• Functional validation by measuring arginine transport using radiolabeled arginine or arginine concentration assays .

Phenotypic Assays Following Knockdown:
The existing literature demonstrates several functional assays that yield meaningful results when combined with SLC7A2 knockdown:

• Proliferation assays using Ki67 immunostaining or CCK-8 .

• Differentiation assessment through MyHC immunofluorescence for myoblast studies .

• Migration and invasion assays using Transwell chambers .

• Protein nitrosylation levels for neuroinflammation studies .

How is SLC7A2 dysregulation implicated in disease pathophysiology?

Research has revealed diverse roles for SLC7A2 across different disease contexts, with seemingly contradictory functions depending on the cellular environment:

Huntington's Disease:
SLC7A2 is selectively upregulated in Huntington's disease (HD) cellular models and patient samples . This upregulation contributes to pathology through:

• Enhanced arginine uptake leading to abnormally high iNOS induction

• Increased NO production resulting in elevated protein nitrosylation

• Exacerbated response to neuroinflammatory challenges

• Potential contribution to mitochondrial dysfunction

These findings suggest SLC7A2 inhibition could be therapeutically beneficial in HD contexts .

Ovarian Cancer:
Conversely, SLC7A2 appears to function as a tumor suppressor in ovarian cancer, where:

• SLC7A2 is significantly downregulated in ovarian cancer samples

• Lower expression correlates with poorer prognosis

• Knockdown promotes cancer cell viability, invasion, and migration

• SLC7A2 loss increases expression of epithelial-mesenchymal transition markers including N-cadherin and vimentin

Other Cancer Types:
Research indicates SLC7A2 expression correlates with survival advantage in breast cancer patients, and loss of SLC7A2 exacerbates inflammation-associated colon tumorigenesis , suggesting context-dependent roles.

This context-dependent functionality underscores the need for careful experimental design when using the SLC7A2-FITC antibody in disease models.

What controls are essential when using the SLC7A2-FITC antibody?

To ensure reliable interpretation of results when using the SLC7A2-FITC antibody, researchers should implement the following controls:

Staining Controls:

• Unstained cells to establish autofluorescence baseline

• Isotype control (rabbit IgG-FITC) to assess non-specific binding

• FMO (Fluorescence Minus One) control when performing multicolor flow cytometry

• Positive control using cells known to express SLC7A2 (e.g., differentiated myoblasts or activated macrophages)

Biological Controls:

• SLC7A2 knockdown cells (using validated siRNA approaches) to confirm staining specificity

• Competitive inhibition using the immunizing peptide ((C)KTYFKMNYTGLAE)

• Treatment controls using conditions known to alter SLC7A2 expression (e.g., differentiation media for myoblasts or inflammatory stimuli for macrophages)

Analysis Controls:

• Time-matched controls when evaluating dynamic changes in SLC7A2 expression

• Standard curves if performing quantitative assessments of expression levels

• Sample preparation controls to account for potential artifacts from cell harvesting procedures

How can researchers integrate SLC7A2-FITC antibody use with functional arginine transport assays?

Integration of SLC7A2 detection with functional arginine transport provides powerful mechanistic insights. Recommended approaches include:

Sequential Analysis Protocol:

• Perform flow cytometry with the SLC7A2-FITC antibody on a portion of cells to quantify surface expression.

• Use the remaining cells for arginine uptake assays by measuring intracellular arginine concentrations.

• Correlate surface expression levels with transport activity across experimental conditions.

Simultaneous Measurement Considerations:

• The antibody binds to amino acids 151-163 in the second extracellular loop , potentially interfering with transport function.

• Design experiments to determine if antibody binding affects transport activity before attempting simultaneous measurements.

• Consider using cell sorting based on SLC7A2-FITC staining intensity followed by functional assays on sorted populations.

Metabolic Consequence Assessment:
Following SLC7A2-FITC-based characterization, researchers can probe downstream metabolic pathways:

• Measure nitric oxide production using fluorescent indicators

• Assess iNOS induction through qPCR or Western blotting

• Quantify protein nitrosylation levels using specialized assays

• Evaluate arginine-derived metabolites through targeted metabolomics

What are the documented relationships between SLC7A2 expression and cellular differentiation states?

The literature reveals important correlations between SLC7A2 expression and cellular differentiation, particularly in myogenic development:

Myoblast Differentiation Pattern:

• SLC7A2 shows gradual upregulation at both mRNA and protein levels during C2C12 myoblast differentiation

• The increase correlates with rising arginine levels, particularly notable on day 4 of differentiation

• Knockdown of SLC7A2 significantly impairs myotube formation and reduces differentiation markers

• Expression of muscle fiber type markers (Myh1, Myh4, Myh7) decreases following SLC7A2 knockdown

This upregulation pattern suggests researchers can use the SLC7A2-FITC antibody as a potential marker for myogenic differentiation progression.

Functional Impact Table: Effects of SLC7A2 Knockdown on Myogenic Markers

These findings highlight the potential utility of the SLC7A2-FITC antibody for monitoring differentiation stages in myogenic research.

How should researchers approach SLC7A2 investigation in neuroinflammatory conditions?

When investigating SLC7A2 in neuroinflammatory conditions such as Huntington's disease, researchers should consider:

Experimental Design Strategy:

• Assess baseline SLC7A2 expression in disease models using the FITC-conjugated antibody via flow cytometry.

• Challenge cells with neuroinflammatory stimuli to observe dynamic changes in SLC7A2 surface expression.

• Correlate expression with functional outcomes such as NO production and protein nitrosylation.

• Implement genetic manipulation (knockdown/knockout) of SLC7A2 to evaluate its causal role in neuroinflammatory response.

Cell Type Considerations:
Research has demonstrated that SLC7A2 upregulation in HD contributes to an overactive response to neuroinflammatory challenges, with experiments conducted in:

• STHdhQ7 and Q111 cell lines (striatal cells)

• Primary mouse astrocytes

• Human patient-derived samples

The FITC-conjugated antibody can be valuable for distinguishing cell-type specific expression patterns in mixed neural cultures or tissue preparations.

Mechanistic Investigation Approach:

• Compare SLC7A2 surface expression with intracellular NO production

• Assess correlation between arginine uptake and iNOS induction

• Measure mitochondrial dynamics in relation to SLC7A2 expression levels

• Evaluate the effects of arginine depletion or supplementation on disease phenotypes

What methodological considerations apply when using SLC7A2-FITC antibody in cancer research?

The search results highlight SLC7A2's divergent roles in cancer, necessitating careful methodological approaches:

Expression Analysis in Clinical Samples:

• The antibody can be used for flow cytometry analysis of dissociated tumor tissue or circulating tumor cells

• Expression patterns should be correlated with clinical parameters including patient age, tumor stage, and survival outcomes

• Comparative analysis across cancer types is warranted given the contrasting roles observed in different cancers

Functional Studies Protocol:

• Establish baseline SLC7A2 expression in cancer cell lines using the FITC-conjugated antibody

• Implement knockdown using validated siRNA approaches

• Assess functional consequences on:

  • Cell viability (using CCK-8 assay)

  • Colony formation capability

  • Invasion and migration (Transwell assays)

  • Epithelial-mesenchymal transition marker expression

  • Response to chemotherapeutic agents such as cisplatin

Notable Research Finding: SLC7A2 knockdown promotes viability, invasion and migration of ovarian cancer cells while having no significant effect on cisplatin sensitivity .

How can SLC7A2-FITC antibody be integrated into multi-parameter analysis workflows?

For comprehensive characterization, researchers can integrate SLC7A2-FITC antibody into multi-parameter workflows:

Flow Cytometry Panel Design:

• Combine SLC7A2-FITC with markers for cell identification, activation status, and functional outcomes

• For myogenic studies: pair with markers of proliferation (Ki67) and differentiation (MyoD, MyHC)

• For cancer research: combine with epithelial-mesenchymal transition markers (E-cadherin, N-cadherin, vimentin)

• For neuroinflammation: integrate with microglial/astrocyte activation markers and oxidative stress indicators

Multi-omics Integration Strategy:

• Sort cells based on SLC7A2-FITC expression levels

• Process sorted populations for:

  • Transcriptomics (RNA-seq) to identify differentially expressed genes

  • Proteomics to assess global protein changes

  • Metabolomics focusing on arginine-related pathways

• Apply computational integration of multi-omics data to identify mechanistic networks

Imaging-Based Approaches:
Though primarily validated for flow cytometry, researchers might adapt the antibody for:

• Imaging flow cytometry to correlate SLC7A2 expression with morphological features

• Live cell imaging to monitor dynamic changes in surface expression

• High-content screening to assess multiple parameters simultaneously in response to perturbations