LIFR Antibody, FITC conjugated

What is LIFR and what biological systems express this receptor?

LIFR (Leukemia Inhibitory Factor Receptor alpha), also known as CD118, is a 185-190 kDa type I transmembrane protein belonging to the Interleukin-6 receptor family. LIFR mediates biological effects of multiple cytokines including Cardiotrophin-1, CLC, CNTF, IL-6, IL-11, IL-27, and Oncostatin M. LIFR expression is widespread across many cell types and tissues, including hepatic sinusoidal endothelium, adrenal cortical cells, uterine epithelium, cardiac muscle cells, embryonic stem cells, odontoblasts, neural precursors, fibroblasts, osteoblasts, megakaryocytes, activated macrophages, and sympathetic neurons . LIFR plays particularly important roles in early pregnancy events such as blastocyst implantation in the uterus and is crucial for embryonic stem cell maintenance and differentiation .

What is the structure and signaling mechanism of LIFR?

The mature mouse LIFR alpha consists of a 785 amino acid extracellular domain (ECD) containing two cytokine receptor homology domains, one WSxWS motif, and three fibronectin type III repeats, followed by a 25 amino acid transmembrane segment and a 239 amino acid cytoplasmic domain . LIFR functions primarily on the cell surface, where it forms a signaling complex with gp130, a shared receptor component for several cytokines . This complex formation is critical for signal transduction that influences cell survival, proliferation, and differentiation. LIFR alone binds LIF with low affinity, but this affinity increases significantly when LIFR associates with gp130 in a ligand-induced manner . The LIFR/gp130 receptor complex also transduces Oncostatin M signals, although LIFR alone does not interact with Oncostatin M .

What are the optimal dilutions and conditions for using LIFR antibody, FITC conjugated in different applications?

• Cell or tissue fixation method (paraformaldehyde vs. methanol)

• Permeabilization requirements (Triton X-100 or saponin for intracellular targets)

• Blocking solutions (typically 10% FBS in PBS)

• Washing steps (usually 3-5 washes with PBS)

• Mounting medium (preferably with anti-fade agents to prevent photobleaching)

Always protect FITC-conjugated antibodies from light exposure during storage and experimental procedures to maintain fluorescence intensity .

How can I validate the specificity of LIFR antibody in my experimental system?

Validating antibody specificity is crucial for generating reliable data. For LIFR antibodies, consider implementing these validation strategies:

• Positive and negative controls: Use cell lines known to express high levels of LIFR (e.g., D3 mouse embryonic stem cells) as positive controls and cell lines with no or low LIFR expression (e.g., BaF3 cells) as negative controls .

• Isotype control: Compare staining with LIFR antibody to an isotype-matched control antibody to assess non-specific binding. For example, when using a LIFR-PE conjugated antibody, compare with an isotype control like IC005P .

• Competitive blocking: Pre-incubate the antibody with recombinant LIFR protein before staining to confirm binding specificity.

• Multiple detection methods: Confirm LIFR expression using complementary techniques such as flow cytometry, western blotting, and immunofluorescence.

• Knockout/knockdown validation: If possible, use LIFR knockout cells or siRNA-mediated knockdown to verify antibody specificity.

The detection of differential LIFR expression in various cell types (e.g., OP9 vs. NIH/3T3 cells) can provide further confidence in antibody specificity .

What controls should be included in flow cytometry experiments using FITC-conjugated LIFR antibodies?

When conducting flow cytometry experiments with FITC-conjugated LIFR antibodies, several controls are essential:

• Unstained cells: To establish baseline autofluorescence and set appropriate voltage settings.

• Isotype control: Use an isotype-matched FITC-conjugated antibody (e.g., IgG1 for monoclonal LIFR antibodies) to assess non-specific binding and establish gating strategies .

• Positive control cells: Include a cell line known to express LIFR at high levels, such as D3 mouse embryonic stem cells .

• Negative control cells: Include cells with minimal or no LIFR expression, such as BaF3 cells .

• Fluorescence minus one (FMO) controls: When performing multi-color flow cytometry, include controls where all fluorophores except FITC are present to properly set compensation.

• Viability dye: Include a viability dye to exclude dead cells, which can bind antibodies non-specifically.

• Blocking controls: In some cases, pre-incubation with Fc receptor blocking reagents may be necessary to reduce non-specific binding, especially when working with immune cells.

How can LIFR antibodies be used to study LIFR-mediated signaling pathways in different cellular contexts?

LIFR antibodies can be powerful tools for investigating LIFR-mediated signaling across various cellular systems:

• Signaling complex formation: Use LIFR antibodies in co-immunoprecipitation experiments to study the interaction between LIFR and gp130, as well as the formation of ternary complexes with other IL-6 family receptors like CNTF receptor alpha .

• Receptor internalization and trafficking: FITC-conjugated LIFR antibodies enable real-time monitoring of receptor internalization following ligand binding using live-cell imaging techniques.

• Phosphorylation cascades: Following LIF stimulation, use LIFR antibodies in combination with phospho-specific antibodies to analyze downstream activation of JAK/STAT, MAPK, and PI3K/AKT pathways by western blotting, immunofluorescence, or flow cytometry.

• Receptor modulation studies: Investigate how LIFR expression changes in response to different stimuli (e.g., cytokines, growth factors) using flow cytometry with FITC-conjugated LIFR antibodies. For example, studies have shown differential LIFR surface expression in OP9 and NIH/3T3 cells after exposure to mouse Oncostatin M (mOSM) .

• Receptor blocking studies: Use LIFR antibodies as blocking agents to inhibit LIF binding and study the functional consequences on cellular processes like differentiation or proliferation.

• Single-cell analysis: Combine FITC-conjugated LIFR antibodies with other phenotypic markers to identify and characterize LIFR-expressing cell subpopulations using multi-parameter flow cytometry.

How do I address inconsistent staining results when using LIFR antibody, FITC conjugated?

Inconsistent staining with FITC-conjugated LIFR antibodies can result from several factors:

• Antibody degradation: FITC is sensitive to light and pH changes. Store antibodies protected from light at 4°C (short-term) or aliquoted at -20°C (long-term) . Avoid repeated freeze-thaw cycles.

• Variable receptor expression: LIFR expression can be dynamically regulated. For example, exposure to 10 ng/mL mOSM can alter LIFR surface expression in a time-dependent manner . Standardize cell culture conditions and treatment protocols.

• Epitope masking: Certain fixation methods may alter the LIFR epitope. Compare different fixation protocols (4% paraformaldehyde vs. methanol) to determine which best preserves antibody recognition.

• Receptor internalization: LIFR can be internalized following ligand binding. Consider performing a time-course experiment after stimulation to capture receptor dynamics.

• Insufficient permeabilization: For intracellular LIFR detection, optimize permeabilization conditions using different detergents (Triton X-100, saponin) at various concentrations.

• Blocking efficiency: Inadequate blocking can lead to high background. Test different blocking solutions (BSA, normal serum, commercial blockers) and durations.

• Antibody concentration: Titrate the antibody to determine the optimal working concentration for your specific application and cell type .

• Buffer composition: Ensure buffer pH is appropriate (typically pH 7.2-7.4) and consider adding protease inhibitors when working with fresh tissue samples.

What are the advantages and limitations of using FITC-conjugated versus PE-conjugated LIFR antibodies?

When choosing between FITC and PE conjugates for LIFR antibodies, consider these comparative aspects:

Advantages of FITC-conjugated LIFR antibodies:

• Smaller molecule size with minimal steric hindrance

• Compatible with many fluorescence microscopy filter sets

• Ideal for multicolor panels with PE/PE-Cy5/PE-Cy7 fluorophores

• More conjugation sites per antibody (higher F/P ratio)

Limitations of FITC-conjugated LIFR antibodies:

• More susceptible to photobleaching, requiring anti-fade reagents

• Lower brightness compared to PE, potentially reducing sensitivity

• Higher background in certain tissues due to autofluorescence

• pH-sensitive (optimal fluorescence at pH 8.0, diminished at lower pH)

Choose FITC-conjugated LIFR antibodies when working with samples with low autofluorescence, when performing multiplexing with PE-conjugated antibodies, or when photobleaching is not a major concern. For applications requiring higher sensitivity or when analyzing samples with significant autofluorescence, PE-conjugated LIFR antibodies may be preferable .

How can FITC-conjugated LIFR antibodies be used to study LIFR's role in embryonic stem cell biology?

LIFR plays a crucial role in maintaining pluripotency in embryonic stem cells (ESCs). FITC-conjugated LIFR antibodies can be employed to investigate this biology in several ways:

• Quantifying LIFR expression during differentiation: Flow cytometry with FITC-conjugated LIFR antibodies can track changes in LIFR expression as ESCs differentiate into various lineages, providing insights into when and how LIFR signaling is regulated during development .

• Sorting LIFR-expressing subpopulations: FACS using FITC-conjugated LIFR antibodies allows isolation of LIFR-high and LIFR-low ESC subpopulations for subsequent functional characterization or transcriptomic analysis.

• Co-localization studies: Combine FITC-conjugated LIFR antibodies with other fluorescently-labeled antibodies against pluripotency markers (Oct4, Nanog, Sox2) to investigate their spatial relationships using confocal microscopy.

• Live imaging of LIFR dynamics: Use FITC-conjugated LIFR antibodies that recognize extracellular epitopes to perform live-cell imaging of receptor dynamics in response to LIF or other cytokines.

• Clonal analysis: Sort single ESCs based on LIFR expression levels and assess their colony-forming efficiency and differentiation potential.

• Receptor internalization and recycling: Track LIFR trafficking in ESCs following LIF stimulation using pulse-chase experiments with FITC-conjugated LIFR antibodies.

When designing these experiments, D3 mouse embryonic stem cells can serve as a positive control for LIFR expression, as demonstrated in flow cytometry analysis .

What are the best approaches for multiplexing FITC-conjugated LIFR antibodies with other fluorescent markers?

Effective multiplexing with FITC-conjugated LIFR antibodies requires careful panel design and instrument setup:

• Fluorophore selection: Combine FITC (green) with fluorophores that have minimal spectral overlap such as PE (orange-red), APC (far-red), and BV421 (violet). Avoid using fluorophores with emission spectra close to FITC (e.g., Alexa Fluor 488).

• Panel design considerations:

  • Assign FITC to high-abundance targets like LIFR when multiplexing with dim fluorophores

  • Place dim fluorophores on highly expressed markers and bright fluorophores on low-expressed markers

  • Account for cellular autofluorescence, which often overlaps with FITC spectrum

• Compensation controls: Prepare single-color controls for each fluorophore in your panel using the same cells or compensation beads. These are essential for accurate compensation, especially between FITC and PE channels where spillover can occur.

• Titration: Titrate each antibody individually before combining them to determine optimal concentrations that provide adequate signal-to-noise ratio.

• Fixation considerations: If fixation is required, ensure the selected fixative preserves fluorescence of all fluorophores in the panel. Paraformaldehyde (1-4%) is generally compatible with most fluorophores.

• Sequential staining: For challenging combinations, consider sequential staining approaches where FITC-conjugated LIFR antibody is applied separately from other antibodies.

• Microscopy-specific considerations: When multiplexing for fluorescence microscopy, acquire images sequentially rather than simultaneously to minimize bleed-through, particularly between FITC and other green-yellow fluorophores.

Example multiplexing panels for flow cytometry:

• FITC-conjugated LIFR + PE-conjugated gp130 + APC-conjugated OSMR

• FITC-conjugated LIFR + PE-Cy7-conjugated CD34 + BV421-conjugated Sca-1 + APC-conjugated c-Kit

How can FITC-conjugated LIFR antibodies be applied to study LIFR dynamics in response to cytokine stimulation?

LIFR expression and localization can change dramatically in response to cytokine stimulation. FITC-conjugated LIFR antibodies are valuable tools for tracking these dynamics:

• Time-course analysis: Perform flow cytometry with FITC-conjugated LIFR antibodies at various time points after cytokine treatment (e.g., 10 ng/mL mOSM) to quantify changes in surface LIFR expression . Research has shown that LIFR surface expression changes significantly in a time-dependent manner after Oncostatin M stimulation in cell lines like OP9 and NIH/3T3 .

• Dose-response relationships: Treat cells with increasing concentrations of cytokines and measure LIFR expression levels by flow cytometry to establish dose-response curves.

• Receptor internalization kinetics: Use time-lapse fluorescence microscopy with FITC-conjugated LIFR antibodies to track receptor internalization following ligand binding. This approach can reveal the rate of endocytosis and intracellular trafficking pathways.

• Receptor recycling studies: Perform pulse-chase experiments with FITC-conjugated LIFR antibodies to distinguish between receptor degradation and recycling pathways after internalization.

• Co-localization with endocytic markers: Combine FITC-conjugated LIFR antibodies with markers for early endosomes (EEA1), late endosomes/lysosomes (LAMP1), or recycling endosomes (Rab11) to track the fate of internalized receptors.

• Signaling correlation: Simultaneously assess LIFR levels and phosphorylation of downstream signaling molecules (STAT3, ERK, AKT) to correlate receptor dynamics with signaling outputs.

• Receptor cross-regulation: Investigate how stimulation with one cytokine affects the expression and responsiveness of LIFR to other cytokines in the IL-6 family.

An experimental approach could involve treating cells with 10 ng/mL of mOSM for different time periods (0, 15, 30, 60, 120 minutes), staining with FITC-conjugated LIFR antibody, and analyzing by flow cytometry to quantify surface expression changes, similar to studies that have demonstrated significant time-dependent changes in LIFR surface expression after cytokine stimulation .

How should I analyze and interpret flow cytometry data generated with FITC-conjugated LIFR antibodies?

Proper analysis of flow cytometry data from FITC-conjugated LIFR antibody staining involves several key considerations:

• Gating strategy:

  • Start with time vs. fluorescence gating to exclude anomalous events

  • Gate on forward/side scatter to identify cells of interest and exclude debris

  • If using a viability dye, gate on live cells only

  • Use isotype control to set positive/negative boundaries for LIFR expression

  • Consider density plots rather than histograms for heterogeneous populations

• Fluorescence parameters to report:

  • Mean Fluorescence Intensity (MFI) for quantitative expression level

  • Percentage of LIFR-positive cells for population analysis

  • MFI ratio (sample MFI / isotype control MFI) for normalized comparisons

• Statistical analysis:

  • For comparing multiple treatments or time points (e.g., LIFR expression after cytokine stimulation), use appropriate statistical tests such as one-way ANOVA with Bonferroni post-test

  • Report p-values and mark significance levels (e.g., * p < 0.05, *** p < 0.001)

  • Include error bars representing standard deviation or standard error

• Visualization recommendations:

  • For bimodal populations, overlay histograms of sample vs. control

  • For comparing multiple conditions, use bar graphs of MFI or percent positive

  • For time-course experiments, plot MFI or percent positive vs. time

• Data normalization approaches:

  • Normalize to unstimulated control when comparing treatments

  • Use relative MFI (rMFI = MFI sample / MFI of reference population)

  • For comparing across experiments, consider using calibration beads

When interpreting results, remember that surface LIFR expression can be dynamically regulated by cytokine stimulation. For example, significant time-dependent changes in LIFR surface expression have been observed after treatment with 10 ng/mL mOSM .

What could cause false positive or false negative results when using FITC-conjugated LIFR antibodies?

Several factors can lead to misleading results when using FITC-conjugated LIFR antibodies:

Potential causes of false positive results:

• Autofluorescence: Certain cell types (particularly macrophages, dendritic cells, and older cells) naturally emit fluorescence in the FITC channel. Solution: Include unstained controls and consider using spectral unmixing or alternative fluorophores.

• Non-specific binding: Fc receptors on immune cells can bind antibodies regardless of specificity. Solution: Use Fc receptor blocking reagents before antibody staining.

• Dead/dying cells: Compromised cell membranes allow antibody entry and non-specific binding. Solution: Include a viability dye and gate on live cells only.

• Inappropriate isotype control: Using an isotype that doesn't match the primary antibody can lead to incorrect gating. Solution: Ensure the isotype control matches the LIFR antibody's isotype (e.g., IgG1 for monoclonal antibodies) .

• Spectral overlap: In multicolor experiments, improper compensation can lead to false FITC signal. Solution: Prepare proper single-color controls and perform accurate compensation.

Potential causes of false negative results:

• Epitope masking: Certain fixation or permeabilization methods may alter the LIFR epitope. Solution: Test multiple fixation protocols or use unfixed cells when possible.

• Receptor internalization: Stimulation with ligands like LIF or OSM can trigger LIFR internalization, reducing surface staining. Solution: Perform staining before stimulation or permeabilize cells for total LIFR detection.

• Low expression levels: LIFR may be expressed at levels near the detection limit. Solution: Use amplification systems or consider more sensitive fluorophores like PE instead of FITC.

• Photobleaching: FITC is particularly susceptible to photobleaching. Solution: Minimize light exposure during staining and acquisition, and use anti-fade reagents.

• Antibody degradation: FITC conjugates can degrade over time, especially with repeated freeze-thaw cycles. Solution: Aliquot antibodies upon receipt and store protected from light at recommended temperatures .

• Competitive binding: High concentrations of soluble LIFR or ligands (LIF) in the sample may compete with antibody binding. Solution: Wash cells thoroughly before staining.

How do I address contradictory data between LIFR protein detection and mRNA expression analysis?

Discrepancies between LIFR protein detection using FITC-conjugated antibodies and mRNA expression can arise from several biological and technical factors:

• Post-transcriptional regulation: mRNA levels may not directly correlate with protein expression due to:

  • MicroRNA-mediated repression of LIFR translation

  • mRNA stability differences affecting transcript half-life

  • Translational efficiency variations

  Solution: Validate findings using multiple protein detection methods (flow cytometry, western blot, immunofluorescence) and consider investigating specific post-transcriptional regulatory mechanisms.

• Protein stability and turnover: LIFR protein may have different degradation rates than its mRNA:

  • Ligand-induced receptor internalization and degradation

  • Proteasomal degradation pathways

  • Cell-type specific differences in protein half-life

  Solution: Perform protein synthesis inhibition (cycloheximide) experiments to assess LIFR protein stability across different conditions.

• Alternative splicing: Different antibodies may recognize specific LIFR isoforms:

  • Soluble LIFR isoform (90 kDa) vs. membrane-bound form (185-190 kDa)

  • Cell-type specific expression of LIFR isoforms

  Solution: Use PCR primers or antibodies that can distinguish between isoforms and verify which form is predominant in your experimental system.

• Technical considerations:

  • Sensitivity differences between protein and mRNA detection methods

  • Antibody specificity issues

  • RNA quality or degradation affecting mRNA measurements

  Solution: Consider absolute quantification methods for both protein (quantitative flow cytometry with calibration beads) and mRNA (digital PCR).

• Experimental design factors:

  • Temporal discrepancies (mRNA changes may precede protein changes)

  • Different detection thresholds for RNA vs. protein methods

  Solution: Perform time-course experiments capturing both mRNA and protein dynamics.

For example, in studies comparing OP9 and NIH/3T3 cells, microarray analysis of Lifr expression showed different patterns than protein-level detection, with protein quantities differing significantly despite similar mRNA levels . This highlights the importance of multi-level analysis when studying receptor expression.