MIA2 Antibody, FITC conjugated

What is the MHC Class II (I-A/I-E) antibody clone M5/114.15.2 and what epitopes does it recognize?

The M5/114.15.2 monoclonal antibody recognizes mouse major histocompatibility complex class II molecules, specifically binding to both I-A and I-E subregion-encoded glycoproteins (I-Ab, I-Ad, I-Aq, I-Ed, I-Ek, but not I-Af, I-Ak, or I-As). It targets a polymorphic determinant present on antigen-presenting cells including B cells, monocytes, macrophages, dendritic cells, and activated T lymphocytes. This antibody is particularly valuable for identifying professional antigen-presenting cells that express MHC class II molecules responsible for presenting exogenous antigens to CD4+ T cells, a critical step in adaptive immune responses.

What are the recommended applications for the FITC-conjugated MHC Class II antibody?

The FITC-conjugated M5/114.15.2 antibody has been validated for several applications:

A single test is defined as the amount of antibody required to stain a cell sample in a final volume of 100 μL, with cell numbers ranging from 10^5 to 10^8 cells per test. For optimal results, researchers should carefully titrate the antibody based on their specific experimental conditions.

How should I optimize antibody concentration for flow cytometry experiments?

For optimal flow cytometry results with the FITC-conjugated MHC Class II antibody, begin with the recommended concentration of 0.25 μg per test, but perform a titration experiment to determine the optimal concentration for your specific cell population. Prepare a serial dilution of the antibody (e.g., 0.5, 0.25, 0.125, 0.0625 μg per test) and stain identical aliquots of your target cells. Analyze the stained samples by measuring both the signal intensity of positive populations and the signal-to-noise ratio. The optimal concentration will provide maximum separation between positive and negative populations while minimizing background fluorescence. Remember that different cell types and sample preparation methods may require different antibody concentrations, so optimization is crucial for each new experimental system.

What controls should be included in experiments using the FITC-conjugated MHC Class II antibody?

A robust experimental design with appropriate controls is essential for accurate interpretation of results:

• Isotype control: Include a FITC-conjugated Rat IgG2b, κ isotype control at the same concentration as your MHC Class II antibody to assess non-specific binding.

• Negative biological control: Use cells known to be negative for MHC Class II expression (e.g., resting T cells) or samples from H-2s or H-2f haplotype mice that should not react with this antibody.

• Positive biological control: Include cells known to express high levels of MHC Class II (e.g., B cells, dendritic cells) from mice with reactive haplotypes.

• Fluorescence-minus-one (FMO) control: Include a sample stained with all fluorochromes in your panel except FITC to properly set gates and account for spectral overlap.

• Compensation controls: When performing multicolor flow cytometry, include single-stained controls for each fluorochrome to properly compensate for spectral overlap.

What is the recommended protocol for antibody conjugation if I need to prepare my own FITC-labeled MHC Class II antibody?

While commercial FITC-conjugated antibodies are readily available, researchers sometimes need to prepare their own conjugates. The process involves:

• Dialyze purified monoclonal antibody against 500 ml FITC labeling buffer (0.1 M sodium carbonate, pH 9.2) at 4°C with 2-3 buffer changes over 2 days, allowing ≥4 hours between changes.

• Determine antibody concentration by measuring absorbance at 280 nm.

• Add 20 μl of freshly prepared 5 mg/ml FITC in anhydrous DMSO for each milligram of antibody and incubate for 2 hours at room temperature.

• Remove unbound FITC by dialysis against PBS with 2-3 buffer changes over 2 days.

• Determine the final concentration and fluorescein/protein (F/P) ratio by measuring absorbance at 280 nm and 495 nm.

An optimal F/P ratio for FITC-antibody conjugates is typically between 2:1 and 6:1. Higher ratios may increase background fluorescence while lower ratios may provide insufficient signal.

Why might I observe weak or no staining of MHC Class II-positive cells?

Several factors can contribute to poor staining results with FITC-conjugated MHC Class II antibody:

• Inappropriate mouse haplotype: Confirm your mouse strain carries a reactive haplotype (H-2b, H-2d, H-2q, H-2p, H-2r, H-2u). Strains with H-2s or H-2f haplotypes will not show reactivity with this antibody.

• Antibody degradation: FITC is sensitive to light and pH changes. Ensure proper storage (protected from light at 4°C) and avoid repeated freeze-thaw cycles.

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

• Cell preparation issues: Overly harsh fixation or permeabilization can damage epitopes. For surface staining, perform antibody incubation before any fixation steps.

• Competition with endogenous ligands: In some experimental conditions, the epitope may be occupied by endogenous proteins. Try using fresher samples or different sample preparation techniques.

How can I reduce background fluorescence when using FITC-conjugated antibodies?

High background fluorescence can significantly impair data quality. To minimize this issue:

• Include blocking step: Pre-incubate cells with 5-10% normal serum (from the same species as the secondary antibody, if applicable) or with Fc receptor blocking reagents.

• Optimize washing steps: Increase the number and volume of washes after antibody incubation to remove unbound antibody.

• Reduce autofluorescence: For tissues with high autofluorescence, consider treatments such as Sudan Black B or commercial autofluorescence reducers.

• Adjust acquisition settings: Use unstained and isotype controls to set proper PMT voltages and compensation.

• Optimize fixation: Some fixatives can increase autofluorescence. If fixation is necessary, try different fixatives or reduce fixation time.

• Consider alternative fluorochromes: If FITC background remains problematic, consider antibodies conjugated to brighter fluorochromes with less spectral overlap with autofluorescence (like PE or APC).

How can I use the MHC Class II antibody to study antigen presentation and T cell activation?

The MHC Class II (I-A/I-E) antibody can be used to investigate the critical process of antigen presentation:

• In vitro antigen presentation assays: Use the antibody to block MHC-II-restricted antigen presentation by pre-treating APCs with the M5/114.15.2 antibody before adding T cells and antigen. This can help determine if T cell responses are MHC-II dependent.

• Visualization of immunological synapses: Combine FITC-conjugated MHC-II antibody with differentially labeled antibodies against T cell receptors or costimulatory molecules to visualize the immunological synapse formation using advanced microscopy techniques.

• Sorting antigen-presenting cell subsets: Use flow cytometry with MHC-II antibody to isolate specific APC populations based on their MHC-II expression levels for downstream functional assays or transcriptomic analysis.

• Monitoring changes in MHC-II expression: Track dynamics of MHC-II expression on APCs during immune responses, inflammation, or following treatment with cytokines or immunomodulators.

What approaches can be used to investigate the role of MHC Class II in immunopeptidomics studies?

Immunopeptidomics involves identifying and characterizing peptides presented by MHC molecules. The M5/114.15.2 antibody can be instrumental in these advanced studies:

• MHC-peptide complex isolation: Use the antibody for immunoprecipitation of MHC-II-peptide complexes from cell lysates, followed by elution of bound peptides.

• Mass spectrometry analysis: After immunoprecipitation with the M5/114.15.2 antibody, eluted peptides can be analyzed by high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to identify specific antigens presented by MHC-II molecules.

• Neoantigen discovery: In cancer immunology research, this approach can identify tumor-specific antigens that might be targets for immunotherapy.

• Validation of predicted epitopes: Combine computational prediction of MHC-II binding peptides with experimental validation using cells expressing the relevant MHC-II haplotypes, identified by M5/114.15.2 staining.

How should I analyze MHC Class II expression levels across different cell populations?

When analyzing MHC Class II expression data from flow cytometry experiments:

• Gating strategy: Establish a hierarchical gating strategy that first identifies viable single cells, then delineates cell populations of interest based on lineage markers before analyzing MHC-II expression.

• Quantitative metrics: Report MHC-II expression using appropriate metrics:

  • Percentage of MHC-II positive cells (using proper isotype controls to set thresholds)

  • Mean or geometric mean fluorescence intensity (MFI or gMFI) to quantify expression levels

  • Molecules of Equivalent Soluble Fluorochrome (MESF) for standardized comparisons across experiments

• Comparative analysis: When comparing MHC-II expression between conditions, use paired statistical tests when appropriate and consider displaying data as fold change relative to control.

• Visualization: Present data as histograms to show distribution of expression within populations, or as dot plots when correlating MHC-II expression with other markers.

What considerations are important when interpreting changes in MHC Class II expression in disease models?

Interpreting MHC Class II expression data in disease models requires careful consideration:

• Cell-specific changes: Distinguish between changes in the proportion of MHC-II+ cells versus changes in expression level per cell. Both have different biological implications.

• Context of immune activation: Interpret MHC-II expression changes in the context of other activation markers, cytokine production, and functional outcomes.

• Strain-specific differences: Remember that different mouse strains have different baseline MHC-II expression levels and different haplotypes that may affect antibody binding.

• Microenvironmental influences: Consider how the tissue microenvironment (inflammation, hypoxia, etc.) might affect MHC-II expression.

• Technical artifacts: Rule out technical causes for apparent changes in expression, such as differences in sample preparation, instrument settings, or antibody lots.

• Biological significance: Correlate changes in MHC-II expression with functional outcomes, such as T cell activation, cytokine production, or disease progression, to establish biological relevance.