LPCAT3 Antibody, FITC conjugated

What is LPCAT3 and what biological role does it play in cellular systems?

LPCAT3 (Lysophosphatidylcholine Acyltransferase 3) is a critical enzyme involved in phospholipid metabolism and membrane remodeling. It primarily functions in the Lands cycle, catalyzing the conversion of lysophosphatidylcholine to phosphatidylcholine through incorporation of acyl-CoA. The enzyme plays essential roles in membrane dynamics, lipid homeostasis, and cellular stress response mechanisms. LPCAT3 has recently gained attention for its involvement in ferroptosis, a form of programmed cell death characterized by iron-dependent lipid peroxidation. Research has demonstrated that LPCAT3 expression is differentially regulated in various cancer types, with particularly notable patterns in melanoma and lung cancer. In experimental settings, researchers typically analyze LPCAT3 through techniques including western blotting, immunohistochemistry, and fluorescence microscopy to understand its expression patterns and subcellular localization.

What are the optimal experimental applications for LPCAT3 antibody (FITC conjugated)?

Based on technical specifications and validation data, LPCAT3 antibody (FITC conjugated) has been optimized for multiple research applications:

The antibody demonstrates specificity for human LPCAT3, particularly targeting specific amino acid regions (e.g., AA 306-363 or AA 122-233 depending on the product) . When designing experiments, researchers should carefully select the antibody based on the target epitope and intended application. The FITC conjugation eliminates the need for secondary antibodies, reducing potential cross-reactivity and simplifying multicolor experimental designs.

What are the critical factors in sample preparation when using LPCAT3 antibody (FITC conjugated)?

Successful detection of LPCAT3 using FITC-conjugated antibodies requires careful attention to sample preparation protocols:

• Fixation protocols: For cellular samples, 4% paraformaldehyde (10-15 minutes) preserves antigen integrity while maintaining fluorescence signal quality. For tissue sections, neutral-buffered formalin fixation followed by paraffin embedding is generally effective.

• Permeabilization requirements: Since LPCAT3 is primarily localized to the endoplasmic reticulum membrane, proper permeabilization is essential. For cell preparations, 0.1-0.5% Triton X-100 (5-10 minutes) typically provides adequate access to the antigen while preserving cellular architecture.

• Antigen retrieval methods: For FFPE tissue sections, heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended to maximize LPCAT3 detection without compromising the FITC fluorophore.

• Blocking conditions: To minimize non-specific binding, samples should be blocked with 5% normal serum (ideally from the same species as the secondary antibody would be, though not needed with direct conjugates) supplemented with 0.1% BSA prior to antibody incubation.

The antibody undergoes purification via antigen-specific affinity chromatography or Protein G purification (>95% purity) , ensuring minimal non-specific binding when proper blocking and washing steps are employed.

How should LPCAT3 antibody (FITC conjugated) be stored and handled to maintain optimal activity?

To preserve antibody integrity and fluorophore activity of LPCAT3 antibody (FITC conjugated):

• Storage temperature: Store at -20°C in aliquots to minimize freeze-thaw cycles . Long-term storage at -80°C may extend shelf life but is typically not necessary.

• Buffer composition: The antibody is typically formulated in 0.01 M PBS, pH 7.4, containing 0.05% Proclin-300 and 50% glycerol as stabilizers . This formulation helps maintain protein stability during freeze-thaw cycles.

• Light sensitivity management: FITC is particularly sensitive to photobleaching. Always protect from light exposure by:

  • Storing in amber tubes or wrapped in aluminum foil

  • Minimizing exposure to ambient light during experimental procedures

  • Working in reduced lighting conditions when possible

• Aliquoting recommendations: Upon receipt, divide the antibody into single-use aliquots (typically 10-20 μL depending on application) to prevent repeated freeze-thaw cycles that can compromise both antibody binding capacity and fluorophore activity.

• Working solution stability: Once diluted to working concentration, the antibody maintains activity for approximately 1-2 weeks when stored at 4°C protected from light.

How can researchers validate the specificity of LPCAT3 antibody (FITC conjugated) in their experimental system?

Comprehensive validation of LPCAT3 antibody (FITC conjugated) requires multiple complementary approaches:

• Positive and negative control samples:

  • Positive controls: Human cell lines with known LPCAT3 expression (e.g., melanocytes, specifically PIG1 cell line)

  • Negative controls: LPCAT3 knockdown or knockout cells generated via siRNA or CRISPR/Cas9 technology

• Competitive inhibition assay: Pre-incubation of the antibody with recombinant LPCAT3 protein (particularly the immunogenic peptide corresponding to AA 306-363) should abolish specific staining.

• Cross-validation with alternative detection methods: Correlate immunofluorescence results with western blot analysis using non-conjugated LPCAT3 antibodies targeting different epitopes.

• Signal pattern assessment: LPCAT3 should demonstrate a characteristic reticular staining pattern consistent with its endoplasmic reticulum localization. Diffuse cytoplasmic or nuclear staining may indicate non-specific binding.

• Quantitative validation: Compare staining intensity across samples with varying LPCAT3 expression levels (e.g., normal versus tumor tissues) to confirm that signal strength correlates with expected biological variation .

How does LPCAT3 expression influence ferroptosis pathways in cancer cells?

LPCAT3 plays a crucial role in regulating ferroptosis susceptibility through its effects on membrane phospholipid composition and lipid peroxidation:

• Mechanistic involvement: LPCAT3 catalyzes the incorporation of polyunsaturated fatty acids (PUFAs) into membrane phospholipids, particularly phosphatidylcholine. These PUFA-containing phospholipids are prime substrates for lipid peroxidation during ferroptosis. Recent research demonstrates that LPCAT3 upregulation promotes lipid peroxidation and ferroptosis in melanoma and lung cancer cells .

• Regulatory pathways: Interferon-γ (IFN-γ) signaling regulates LPCAT3 expression through the STAT1-IRF1 pathway. Treatment with mefloquine (Mef) enhances this signaling cascade, resulting in increased LPCAT3 expression and heightened ferroptosis susceptibility . Knockdown of LPCAT3 inhibits the induction of reactive oxygen species (ROS) and lipid peroxidation by Mef+IFN-γ in cancer cells, confirming its direct involvement in the ferroptotic process .

• Experimental approaches to investigate LPCAT3-mediated ferroptosis:

  • Combine LPCAT3 immunofluorescence with lipid peroxidation assays (C11-BODIPY) to correlate expression with ferroptosis markers at the single-cell level

  • Monitor changes in LPCAT3 expression in response to ferroptosis inducers (e.g., RSL3, erastin, Mef) using flow cytometry with FITC-conjugated LPCAT3 antibody

  • Assess ferroptosis outcomes following genetic manipulation of LPCAT3 levels

• Clinical relevance: LPCAT3 expression is generally lower in tumors than in normal tissues , suggesting that reduced LPCAT3 levels may contribute to ferroptosis resistance in cancer cells. Therapeutic approaches aimed at restoring LPCAT3 expression or function could potentially sensitize cancers to ferroptosis-inducing treatments.

What methodological approaches can optimize LPCAT3 antibody (FITC conjugated) for multicolor flow cytometry?

Optimizing LPCAT3 antibody (FITC conjugated) for multicolor flow cytometry requires strategic panel design and careful technical considerations:

• Spectral compatibility planning:

  • FITC profile: Excitation/emission maxima at 499/515 nm; compatible with standard 488 nm laser

  • Avoid fluorophores with substantial spectral overlap (PE, GFP)

  • Recommended compatible fluorophores: APC (640/660 nm), PerCP (482/678 nm), and BV421 (407/421 nm)

• Intracellular staining protocol optimization:

  • Fixation: 2-4% paraformaldehyde (10-15 minutes) preserves cellular architecture

  • Permeabilization: 0.1% saponin or 0.1-0.3% Triton X-100 for adequate antibody access to intracellular LPCAT3

  • Blocking: 2-5% normal serum in permeabilization buffer (30 minutes) reduces background

• Essential controls for accurate analysis:

  • Single-stain compensation controls for each fluorochrome

  • Fluorescence Minus One (FMO) control specifically excluding LPCAT3-FITC

  • Isotype control (rabbit IgG-FITC) at matched concentration

  • Biological controls: LPCAT3 knockdown cells and positive control cells with known LPCAT3 expression

• Analysis considerations:

  • Sequential gating strategy: Forward/side scatter → single cells → live cells → LPCAT3+ cells

  • Consider correlation with markers of cell state (proliferation, apoptosis) or cell type-specific markers

  • For heterogeneous samples, use co-staining with lineage markers to identify specific populations

• Technical troubleshooting:

  • Signal-to-noise ratio optimization through titration (starting with 5-20 μg/ml)

  • Minimize protocol duration to prevent signal degradation

  • Consider fixation post-staining to preserve signal for downstream analysis

How can LPCAT3 antibody (FITC conjugated) be utilized to investigate the relationship between LPCAT3 expression and anti-PD-1 immunotherapy response?

Recent research has established a significant correlation between LPCAT3 expression and response to anti-PD-1 immunotherapy, offering several strategic approaches for investigation:

• Clinical correlation studies:

  • LPCAT3 expression analysis in patient-derived specimens reveals higher expression in anti-PD-1 responsive groups (complete response + partial response) compared to resistant (progressive disease) patients

  • Analysis of public datasets shows positive correlation between LPCAT3 expression and infiltration of cytotoxic T cells, suggesting its potential role in immunotherapy response

• Experimental methodologies:

  • Flow cytometry using LPCAT3-FITC antibody to quantify expression levels in tumor specimens before and during immunotherapy

  • Multiplex immunofluorescence combining LPCAT3-FITC with immune markers (CD8, PD-1, PD-L1) to assess spatial relationships

  • Single-cell analysis correlating LPCAT3 expression with immunotherapy response markers

• Mechanism investigation approaches:

  • Monitor changes in LPCAT3 expression following IFN-γ treatment using flow cytometry or fluorescence microscopy

  • Assess how LPCAT3 modulation affects lipid peroxidation and ferroptosis in tumor cells following exposure to activated T cells

  • Combinatorial studies evaluating LPCAT3 expression, ferroptosis markers, and T cell activity in response to mefloquine and anti-PD-1 therapy

• Translational research applications:

  • LPCAT3 expression profiling as a potential predictive biomarker for anti-PD-1 therapy response

  • Testing whether pharmacological upregulation of LPCAT3 (e.g., with mefloquine) enhances efficacy of immunotherapy

  • Evaluation of LPCAT3 in combination with established biomarkers (PD-L1, tumor mutational burden) for improved patient stratification

Single-cell RNA sequencing analysis has revealed reduced LPCAT3 expression specifically in melanoma cells from patients resistant to anti-PD-1 treatment , suggesting cell type-specific regulation that warrants further investigation using the cellular resolution provided by FITC-conjugated LPCAT3 antibody.

What are the optimal parameters for using LPCAT3 antibody (FITC conjugated) in confocal microscopy?

Obtaining high-quality confocal microscopy data with LPCAT3 antibody (FITC conjugated) requires optimization of multiple technical parameters:

• Sample preparation protocol refinement:

  • Fixation: 4% paraformaldehyde (10-15 minutes at room temperature) preserves cellular architecture while maintaining antigen accessibility

  • Permeabilization: 0.2% Triton X-100 (5-7 minutes) for balanced membrane permeation

  • Blocking: 5% normal goat serum with 1% BSA (1 hour at room temperature) to minimize background

  • Antibody incubation: 5-20 μg/ml in blocking buffer (overnight at 4°C)

• Imaging parameter optimization:

  • Excitation: 488 nm laser line at 10-15% power to minimize photobleaching

  • Emission collection: 505-530 nm bandpass filter

  • Pinhole setting: 1 Airy unit for optimal balance between resolution and signal strength

  • Scan speed: Slow scanning (4-8 μs pixel dwell time) to improve signal-to-noise ratio

  • Line averaging: 2-4 line averages to reduce noise without excessive photobleaching

• Multiple labeling considerations:

  • For colocalization with ER markers: Use far-red fluorophores (e.g., Alexa 647) to minimize spectral overlap

  • Nuclear counterstain: DAPI (405 nm excitation) works well with FITC without significant bleed-through

  • Sequential scanning recommended to prevent crosstalk between channels

  • For multicolor imaging, acquire FITC channel first due to potential photobleaching

• Image processing and analysis approaches:

  • Deconvolution algorithms improve resolution and signal-to-noise ratio

  • Colocalization analysis with organelle markers (e.g., calnexin for ER) to confirm expected subcellular localization

  • Z-stack acquisition (0.3-0.5 μm steps) for three-dimensional visualization of LPCAT3 distribution

  • Quantitative intensity analysis to compare expression levels between experimental conditions

How can researchers investigate LPCAT3's role in the IFN-γ-STAT1-IRF1 signaling axis using FITC-conjugated antibodies?

The role of LPCAT3 in the IFN-γ-STAT1-IRF1 signaling cascade can be effectively investigated using integrated approaches:

• Temporal regulation analysis:

  • Time-course experiments tracking IFN-γ treatment effects on LPCAT3 expression using FITC-conjugated antibody with flow cytometry or fluorescence microscopy

  • Western blot analysis of STAT1 phosphorylation and IRF1 upregulation in parallel with LPCAT3 expression changes

  • Real-time monitoring of signaling dynamics using live-cell imaging with LPCAT3 reporter constructs

• Mechanistic pathway dissection:

  • Chromatin immunoprecipitation (ChIP) assays to confirm IRF1 binding to the LPCAT3 promoter region following IFN-γ stimulation

  • siRNA-mediated knockdown of STAT1 or IRF1 to establish dependency of LPCAT3 upregulation on these transcription factors

  • Luciferase reporter assays with wild-type and mutated LPCAT3 promoter constructs to identify critical regulatory elements

• Advanced imaging approaches:

  • Co-immunofluorescence with LPCAT3-FITC antibody and antibodies against phospho-STAT1 and IRF1

  • Proximity ligation assays to detect potential protein-protein interactions

  • FRET-based approaches to monitor protein interactions in living cells

• Functional correlation studies:

  • Combined analysis of LPCAT3 expression, lipid peroxidation markers, and ferroptosis outcomes following pathway manipulation

  • Assessment of how mefloquine enhances IFN-γ-induced LPCAT3 expression through STAT1-IRF1 signaling

  • Correlation of pathway activation with anti-PD-1 immunotherapy response in experimental models

Research demonstrates that mefloquine enhances LPCAT3 expression through augmentation of IFN-γ-induced STAT1-IRF1 signaling, which subsequently promotes ferroptosis in melanoma and lung cancer cells . This mechanism represents a promising approach to enhance immunotherapy efficacy that can be further explored using LPCAT3 antibodies.

What are the methodological approaches for analyzing differential LPCAT3 expression between normal and cancerous tissues?

Analysis of LPCAT3 expression differences between normal and malignant tissues requires rigorous methodological approaches:

• Tissue sample preparation strategies:

  • Paired normal-tumor specimens from the same patient to minimize individual variation

  • Tissue microarrays for high-throughput analysis across multiple patients

  • Fresh-frozen samples for optimal protein preservation

  • FFPE sections with optimized antigen retrieval for retrospective studies

• Quantitative assessment techniques:

  • Digital pathology approaches:

    • Whole slide scanning of LPCAT3-stained sections

    • Computer-assisted quantification using machine learning algorithms

    • Multi-region analysis to account for tumor heterogeneity

  • Flow cytometry with LPCAT3-FITC:

    • Single-cell resolution assessment of expression levels

    • Combination with cell type-specific markers

    • Sorting of LPCAT3-high versus LPCAT3-low populations for functional studies

  • Molecular profiling methods:

    • Western blotting for semi-quantitative protein assessment

    • qRT-PCR for mRNA expression analysis

    • Single-cell RNA sequencing for cell type-specific expression patterns

• Data analysis and interpretation:

  • Statistical approaches for comparing expression levels

  • Correlation with clinical parameters (stage, grade, outcome)

  • Integration with other molecular markers (e.g., IRF1, IFN-γ, CD8a)

Clinical data analysis has revealed that LPCAT3 expression is generally lower in tumors compared to normal tissues, with particularly notable reductions in melanoma cell lines compared to normal melanocytes . Single-cell RNA sequencing analysis further demonstrates reduced LPCAT3 expression specifically in melanoma cells from patients resistant to anti-PD-1 treatment , highlighting its potential significance as a biomarker and therapeutic target.

How can researchers correlate LPCAT3 expression with lipid peroxidation and ferroptosis in experimental models?

Investigating the relationship between LPCAT3, lipid peroxidation, and ferroptosis requires integrated methodological approaches:

• Comprehensive assessment techniques:

  • Dual fluorescence analysis:

    • LPCAT3-FITC antibody combined with C11-BODIPY (lipid peroxidation sensor)

    • Flow cytometry or confocal microscopy for single-cell correlation

    • Time-lapse imaging to track temporal relationship between LPCAT3 upregulation and peroxidation onset

  • Biochemical assays:

    • Malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE) quantification as lipid peroxidation markers

    • Glutathione depletion measurement to assess ferroptosis progression

    • Iron chelation rescue experiments to confirm ferroptotic cell death mechanism

• Genetic manipulation approaches:

  • LPCAT3 knockdown using siRNA or shRNA approaches inhibits ROS and lipid peroxidation induced by mefloquine+IFN-γ treatment

  • CRISPR-Cas9-mediated knockout to establish necessity of LPCAT3 for ferroptosis

  • Overexpression studies to determine sufficiency of LPCAT3 in sensitizing cells to ferroptotic stimuli

• Pharmacological intervention studies:

  • Treatment with mefloquine combined with IFN-γ enhances LPCAT3 expression and ferroptosis

  • Ferroptosis inhibitors (ferrostatin-1, liproxstatin-1) to establish specificity

  • Lipid peroxidation inhibitors (vitamin E) to determine mechanistic dependency

• Translational research approaches:

  • Ex vivo analysis of patient-derived samples for LPCAT3 expression and ferroptosis markers

  • In vivo models examining how LPCAT3 modulation affects tumor growth and response to immunotherapy

  • Correlation of LPCAT3 expression with ferroptosis gene signatures in clinical datasets