MHC Class II, I-A Antibody, FITC
Product Specs
Q&A
What is MHC Class II (I-A/I-E) Antibody and what cellular structures does it recognize?
MHC Class II molecules are heterodimeric transmembrane glycoproteins expressed on the surface of antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells. The M5/114.15.2 antibody specifically recognizes both I-A and I-E subregion-encoded Ia glycoproteins in mice, including I-Ab, I-Ad, I-Aq, I-Ed, and I-Ek . MHC Class II consists of two transmembrane proteins: a 35 kDa α (heavy) chain and a 29 kDa β (light) chain. The N-terminal α1 and β1 domains form the antigen-binding groove, which is critical for presenting processed antigens to CD4+ T cells .
What are the specific haplotype reactivities of the M5/114.15.2 clone?
The M5/114.15.2 antibody detects polymorphic determinants present on cells from mice carrying the H-2b, H-2d, H-2q, H-2p, H-2r, and H-2u haplotypes. Importantly, it does not react with cells from mice carrying the H-2s or H-2f haplotypes . This specificity is crucial when designing experiments with different mouse strains. The antibody has been reported to inhibit I-A subregion-restricted T cell responses of the H-2b, H-2d, H-2q, and H-2u haplotypes, but not the H-2f, H-2k, or H-2s haplotypes .
What are the recommended storage conditions to maintain optimal antibody performance?
For FITC-conjugated MHC Class II antibodies, the following storage conditions are recommended:
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Protect from light exposure to prevent photobleaching of the FITC fluorophore
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Store in the provided buffer, typically PBS with 0.09% sodium azide
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When stored properly, the antibody remains stable for one year after shipment
Improper storage can lead to decreased signal intensity and increased background in flow cytometry and immunofluorescence applications.
What applications are validated for MHC Class II, I-A/I-E FITC antibodies?
According to the search results, this antibody has been validated for several research applications:
The antibody has been specifically tested in mouse splenocytes and consistently shows positive detection in flow cytometry applications .
What are the spectral characteristics of FITC conjugates for experimental planning?
The FITC fluorophore conjugated to this antibody has specific spectral properties that researchers should consider when designing multi-color experiments:
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Excitation maximum: 495 nm (optimally excited by blue lasers at 490 nm)
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Compatible with standard FITC filter sets on flow cytometers and fluorescence microscopes
These spectral characteristics are important when planning multicolor panels to avoid spectral overlap with other fluorophores.
How can staining protocols be optimized for MHC Class II (I-A/I-E) FITC in flow cytometry?
For optimal staining results with MHC Class II (I-A/I-E) FITC antibody in flow cytometry:
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Titrate the antibody for each experimental system to determine optimal concentration. While 0.25 μg per 10^6 cells is recommended, this should be verified for each specific application .
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When analyzing professional APCs (dendritic cells, macrophages, B cells), lower antibody concentrations may be sufficient due to high expression levels. For cells with variable expression (activated T cells, IECs), higher concentrations might be needed.
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Include appropriate blocking steps using Fc block before staining to prevent non-specific binding.
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When analyzing tissues with high autofluorescence (such as intestinal tissues), consider using fluorescence-minus-one (FMO) controls to accurately set gates.
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Sample-dependent optimization is critical - check validation data in reference galleries for similar experimental conditions .
What is the significance of MHC Class II expression on intestinal epithelial cells compared to professional APCs?
Intestinal epithelial cells (IECs) express high levels of MHC Class II molecules but are not considered canonical antigen-presenting cells . Recent research indicates that:
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IEC-intrinsic MHC Class II expression plays a role in responses toward the microbiota. Mice with IEC-intrinsic deletion of MHC Class II (IEC^ΔMHC class II) have fewer microbial-bound IgA, fewer regulatory T cells (Tregs), and altered immune repertoire selection .
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While intestinal mononuclear phagocytes (MNPs) have similar MHC Class II transcription levels as IECs, they display less surface MHC Class II and can acquire MHC Class II molecules from IECs through cell-to-cell transfer .
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This epithelial-myeloid exchange of MHC Class II appears to constrain immunity and influence gut homeostasis, suggesting a non-canonical role for MHC Class II in intestinal epithelial cells .
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When staining for MHC Class II in intestinal tissues, researchers should account for this epithelial expression pattern and consider using additional markers to distinguish between epithelial and immune cell populations.
What controls should be included when using MHC Class II (I-A/I-E) FITC antibody?
To ensure reliable and reproducible results with MHC Class II (I-A/I-E) FITC antibody, incorporate the following controls:
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Isotype control: Use a rat IgG2b kappa isotype control conjugated to FITC at the same concentration as the MHC Class II antibody to assess non-specific binding.
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Haplotype controls: Include both positive (H-2b, H-2d, H-2q) and negative (H-2s, H-2f) haplotype samples when possible to confirm specificity .
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Fluorescence-minus-one (FMO) control: Include all antibodies in your panel except MHC Class II-FITC to properly set gates, especially in multicolor experiments.
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Blocking controls: Test the effect of pre-incubating cells with unlabeled M5/114.15.2 antibody before adding the FITC-conjugated version to confirm specificity.
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Biological controls: Include samples with known upregulation (e.g., IFN-γ stimulated cells) and downregulation of MHC Class II to verify antibody sensitivity.
How do fixation methods affect MHC Class II epitope recognition by the M5/114.15.2 clone?
Fixation can significantly impact the detection of MHC Class II by the M5/114.15.2 clone:
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For fixed/permeabilized samples, the recommended dilution (1:200) may differ from that used for live cell staining . This suggests that fixation affects antibody binding kinetics or epitope accessibility.
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Paraformaldehyde fixation (typically 2-4%) generally preserves MHC Class II epitopes recognized by the M5/114.15.2 clone, making it suitable for most applications.
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Methanol-based fixation should be avoided as it can denature MHC Class II molecules and destroy the conformational epitope recognized by the M5/114.15.2 antibody.
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For optimal results in immunofluorescence with frozen sections, a dilution range of 1:200-1:800 is recommended , but researchers should perform titration with their specific fixation protocol.
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When fixation is necessary, perform a side-by-side comparison of fixed versus unfixed samples to assess any impact on staining intensity or pattern.
How can MHC Class II (I-A/I-E) FITC antibodies be incorporated into multi-parameter flow cytometry panels?
When incorporating MHC Class II (I-A/I-E) FITC into multi-parameter panels:
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Consider the brightness of FITC (medium intensity) when assigning markers to fluorophores. Reserve brighter fluorophores (PE, APC) for markers with lower expression.
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Account for FITC's spectral properties (excitation: 495 nm, emission: 524 nm) when selecting other fluorophores to minimize spillover.
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FITC has considerable spectral overlap with PE, so careful compensation is required when using both fluorophores.
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When analyzing dendritic cell or macrophage subsets, combine MHC Class II-FITC with lineage markers (CD11c, F4/80) and activation markers (CD80, CD86) to assess functional states.
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For intestinal tissue analysis, consider using epithelial markers (EpCAM) alongside MHC Class II to distinguish between epithelial and professional APC expression.
What functional impacts should be considered when using MHC Class II antibodies in experimental systems?
The M5/114.15.2 mAb is reported to inhibit I-A subregion-restricted T cell responses in specific haplotypes , which has important implications for functional studies:
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In antigen presentation assays, pre-incubation with unlabeled antibody may block T cell activation. Consider timing of antibody addition in relation to functional readouts.
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For in vitro systems where MHC Class II function is being studied, use the antibody for phenotyping separate aliquots of cells rather than cells intended for functional assays.
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In epithelial-immune cell co-culture systems, the antibody may interfere with the epithelial-myeloid exchange of MHC Class II molecules described in recent literature .
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When interpreting results from studies where cells were exposed to the antibody, consider potential blocking effects that might influence downstream T cell responses.
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For in vivo applications, be aware that antibody administration could potentially modify immune responses by blocking antigen presentation.
How can researchers assess the transfer of MHC Class II molecules between cell types?
Recent research has shown that MHC Class II can be transferred from intestinal epithelial cells to mononuclear phagocytes . To study this phenomenon:
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Fluorescence microscopy: Use the MHC Class II-FITC antibody in combination with cell-specific markers to visualize potential colocalization or transfer between cell types.
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Flow cytometry-based approaches:
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Label one cell population (e.g., epithelial cells) with a stable dye
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Co-culture with unlabeled recipient cells
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Assess MHC Class II expression on recipient cells over time using the FITC-conjugated antibody
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Genetic approaches: Use mice with cell-type specific expression of fluorescently tagged MHC Class II molecules to track intercellular transfer.
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Transwell experiments: Determine if direct cell contact is required for MHC Class II transfer by separating cell populations with a permeable membrane.
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Ex vivo tissue imaging: Examine tissue sections for evidence of MHC Class II transfer at epithelial-immune cell interfaces using confocal microscopy.
What are common pitfalls when using MHC Class II antibodies for identifying dendritic cell subsets?
When using MHC Class II antibodies to identify dendritic cell (DC) subsets, researchers should be aware of:
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Varying MHC Class II expression levels across DC subsets - conventional DC1s (cDC1s) typically express lower levels than cDC2s, requiring careful gating strategies.
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Macrophages and B cells also express high levels of MHC Class II, necessitating additional lineage markers to ensure proper identification of DC subsets.
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Activation state significantly impacts MHC Class II expression levels on DCs, potentially leading to misclassification of activated versus resting populations.
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Tissue-specific differences in MHC Class II expression - DCs from different anatomical locations may require different staining protocols and antibody concentrations.
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The specificity of the M5/114.15.2 clone for certain mouse haplotypes means that strain selection is critical for experimental design.
How does inflammation alter MHC Class II expression, and how should antibody concentration be adjusted?
Inflammation significantly impacts MHC Class II expression patterns:
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Pro-inflammatory cytokines, particularly IFN-γ, upregulate MHC Class II expression on professional APCs and can induce expression on non-classical APCs.
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In inflamed tissues, consider using lower antibody concentrations (1:300-1:400) to prevent saturation and high background.
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Include unstimulated control samples to establish baseline expression levels and optimize detection of inflammation-induced changes.
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When comparing inflamed and non-inflamed tissues, maintain consistent antibody concentrations across samples and use mean fluorescence intensity (MFI) rather than percent positive cells as a more quantitative measure.
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In models of chronic inflammation, evaluate whether MHC Class II expression stability correlates with disease progression or resolution.
What validation techniques should be employed when studying tissues with variable MHC Class II expression?
For tissues with heterogeneous MHC Class II expression:
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Immunohistochemistry validation: Compare staining patterns in tissue sections to flow cytometry results to confirm cellular distribution.
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Genetic validation: Use MHC Class II knockout tissues or cells as negative controls to confirm specificity.
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Multi-technique validation: Confirm flow cytometry findings with western blot or RT-PCR analysis of MHC Class II levels.
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Cell sorting validation: Sort MHC Class II-positive and negative populations and reanalyze to confirm separation and expression levels.
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Biological validation: Stimulate cells with IFN-γ to upregulate MHC Class II and confirm increased staining, serving as a positive control for antibody sensitivity.
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Cross-antibody validation: When possible, compare results using alternative clones recognizing different epitopes on MHC Class II molecules.
Product Science Overview
The MHC Class II, (I-A) (Mouse Antibody, FITC) is a monoclonal antibody that targets the major histocompatibility complex (MHC) class II molecules, specifically the I-A and I-E subregion-encoded glycoproteins in mice. This antibody is conjugated with fluorescein isothiocyanate (FITC), a fluorescent dye, which allows for the visualization and analysis of MHC class II molecules using flow cytometry and other fluorescence-based techniques.
MHC class II molecules are essential for the immune system’s ability to recognize and respond to foreign antigens. They are primarily expressed on antigen-presenting cells (APCs) such as B cells, dendritic cells, macrophages, and a subset of activated T cells. These molecules present processed antigenic peptides to CD4+ T helper cells, initiating an immune response.
The MHC Class II, (I-A) (Mouse Antibody, FITC) specifically reacts with a polymorphic determinant shared by the I-A^b, I-A^d, I-A^q, I-E^d, and I-E^k MHC class II alloantigens. It does not react with I-A^f, I-A^k, or I-A^s alloantigens . This specificity makes it a valuable tool for studying the expression and function of MHC class II molecules in various mouse strains.
This antibody is widely used in immunological research for various applications, including:
- Flow Cytometry: The FITC conjugation allows for the detection and quantification of MHC class II molecules on the surface of cells using flow cytometry .
- Immunohistochemistry: It can be used to stain tissue sections to visualize the distribution of MHC class II molecules in different tissues .
- Immunoprecipitation: The antibody can be used to isolate MHC class II molecules from cell lysates for further analysis .
- Blocking Studies: It can be used to block MHC class II-mediated antigen presentation, helping to study the role of these molecules in immune responses .
The MHC Class II, (I-A) (Mouse Antibody, FITC) is typically purified by affinity chromatography and conjugated with FITC under optimal conditions. The antibody solution is formulated in a phosphate-buffered solution (pH 7.2) containing 0.09% sodium azide as a preservative. It should be stored undiluted between 2°C and 8°C and protected from prolonged exposure to light to maintain its stability and functionality .
