MHC CLASS II, I-E Antibody, FITC

What is MHC Class II I-A/I-E and why is it important in immunological research?

MHC Class II molecules, including I-A and I-E, are essential glycoproteins expressed on antigen-presenting cells that play a crucial role in the adaptive immune response. These molecules present antigenic peptides to CD4+ T cells, facilitating immune recognition and response. MHC Class II is fundamental to understanding antigen presentation, T cell activation, and immune regulation mechanisms. These molecules are expressed primarily on professional antigen-presenting cells (APCs) including B cells, monocytes, macrophages, dendritic cells, and activated T lymphocytes . Their significant function in immune regulation makes them valuable targets for research into autoimmune disorders, chronic inflammatory conditions, and infectious diseases .

What is the specificity of the M5/114.15.2 clone for MHC Class II detection?

The M5/114.15.2 monoclonal antibody recognizes a shared extracellular determinant on multiple mouse MHC Class II molecules, specifically I-Ab, I-Ad, I-Aq, I-Ed, and I-Ek alloantigens. This antibody does not react with I-Af, I-Ak, or I-As . The antibody can detect MHC Class II expression on cells from mice carrying the H-2b, H-2d, H-2q, H-2p, H-2r, and H-2u haplotypes, but not from mice carrying the H-2s or H-2f haplotypes . Additionally, M5/114 mAb is reported to inhibit I-A-restricted T cell responses of the H-2b, H-2d, H-2q, and H-2u haplotypes, but not H-2f, H-2k, or H-2s haplotypes .

What are the primary applications for FITC-conjugated MHC Class II antibodies?

FITC-conjugated MHC Class II antibodies are primarily utilized in:

• Flow cytometry (both live and fixed/permeabilized cells)

• Immunofluorescence (particularly on frozen sections)

These applications allow researchers to detect and quantify MHC Class II expression on various cell populations. The FITC conjugation provides bright green fluorescence, enabling precise detection in fluorescence-based assays .

For optimal flow cytometry results, approximately 0.25 μg of antibody per test is typically recommended .

How can MHC Class II expression patterns be used to identify specific antigen-presenting cell subpopulations?

Professional antigen-presenting cells show differential levels of MHC Class II expression, which can be leveraged for identification and characterization. Using FITC-conjugated MHC Class II antibodies in multi-parameter flow cytometry:

• Dendritic cells and B cells exhibit the highest levels of MHC Class II expression, showing strong co-expression with CD74 (invariant chain)

• Monocytes typically show intermediate MHC Class II expression and may be weakly immunoreactive for I-A/I-E

• Macrophages of protective/suppressive haplotypes express MHC Class II at higher levels compared to disease-associated haplotypes, which correlates with their capacity to exert Th1 bias

This differential expression pattern allows researchers to distinguish between various APC populations in heterogeneous samples and correlate MHC Class II expression levels with functional properties in immune responses.

What considerations should be made when analyzing MHC Class II expression in inflammatory disease models?

When studying inflammatory conditions using MHC Class II expression:

• Consider haplotype-specific effects: Different mouse strains express different MHC haplotypes that may influence disease susceptibility. The level of expression of MHC Class II molecules might "gate" the back signal from T cells to APCs, where heightened expression could increase IL-12 signaling and exert a protective/suppressive effect via Th1 bias .

• Evaluate expression dynamics: MHC Class II expression is upregulated during inflammation, particularly in response to IFN-γ. Reporter mice expressing fluorescent proteins under MHC Class II component control (e.g., Cd74-tdTomato) have demonstrated that the level of reporter expression correlates well with MHC Class II receptor expression .

• Consider cell type-specific responses: In inflammatory models, traditionally non-APCs such as oligodendroglia can upregulate MHC Class II components in response to inflammatory stimuli, as demonstrated in mouse models and human MS brain tissue .

• Account for microenvironmental factors: Local cytokine milieu significantly affects MHC Class II expression patterns and subsequent T cell polarization (Th1 vs. Th2) .

How do MHC Class II reporter systems compare with antibody-based detection for studying antigen presentation dynamics?

MHC Class II reporter systems (such as Cd74-tdTomato mice) offer distinct advantages and limitations compared to antibody-based detection:

Advantages of reporter systems:

• Enable live-cell imaging and longitudinal tracking

• Allow visualization of MHC Class II component expression in tissues without antibody penetration issues

• Can report transcriptional activation of MHC components

Limitations of reporter systems:

• May not perfectly correlate with surface expression of assembled MHC Class II complexes

• Reporter signal may persist longer than the actual protein

Researchers should select the appropriate system based on experimental needs, potentially using both approaches for comprehensive analysis of antigen presentation dynamics.

What protocol optimizations are recommended for detecting MHC Class II using FITC-conjugated antibodies in flow cytometry?

For optimal detection of MHC Class II using FITC-conjugated antibodies in flow cytometry:

• Sample preparation:

  • For splenocytes or other lymphoid tissues: Prepare single-cell suspensions and filter to remove cell clumps

  • Use appropriate blocking buffer (containing 1-2% BSA or FBS) to reduce non-specific binding

• Staining protocol:

  • Recommended antibody concentration: 0.25 μg per test or dilution of 1:200 (approximately 2-3 μg/ml)

  • Incubation time: 20-30 minutes at 4°C in the dark

  • Include appropriate isotype control: FITC Rat IgG2b, κ

• Instrument settings:

  • FITC is optimally excited by the blue (488 nm) laser

  • Collect emission in the standard FITC channel (typically 525/20 nm bandpass filter)

  • Perform proper compensation if using multiple fluorochromes

• Analysis considerations:

  • When analyzing heterogeneous populations, use additional markers to identify specific cell types

  • Consider using viability dyes to exclude dead cells, which can bind antibodies non-specifically

What are effective strategies for troubleshooting weak or inconsistent MHC Class II staining?

When encountering weak or inconsistent MHC Class II staining:

• Verify haplotype compatibility:

  • Confirm your mouse strain expresses MHC haplotypes recognized by the M5/114.15.2 clone (H-2b, H-2d, H-2q, H-2p, H-2r, H-2u)

  • The antibody does not react with H-2s or H-2f haplotypes

• Optimize antibody concentration:

  • Titrate the antibody to determine optimal concentration for your specific application

  • Generally start with recommended concentration (2-3 μg/ml) and adjust as needed

• Improve signal-to-noise ratio:

  • Ensure proper blocking to reduce background

  • Protect FITC-conjugated antibodies from prolonged light exposure during storage and staining

  • Store at 2-8°C and do not freeze

• Consider activation status:

  • MHC Class II expression varies with cellular activation state

  • B cells show increased expression after LPS activation

  • Macrophages show differential expression based on activation state (bone marrow-derived vs. splenic)

• Check reagent quality:

  • FITC is sensitive to photobleaching; minimize light exposure

  • Verify antibody viability with positive control samples

How can FITC-conjugated MHC Class II antibodies be effectively used in multi-parameter experimental designs?

For multi-parameter experiments incorporating FITC-conjugated MHC Class II antibodies:

• Panel design considerations:

  • FITC has moderate brightness and is excited by the 488 nm laser

  • Reserve brighter fluorochromes (PE, APC) for lower-abundance targets

  • Consider spillover: FITC has significant spectral overlap with PE that requires proper compensation

• Compatibility with reporter systems:

  • When using with fluorescent reporter mice (e.g., Cd74-tdTomato), select fluorochromes with minimal spectral overlap with tdTomato

  • When combining with reporter systems, validate correlation between antibody staining and reporter signal

• Functional correlation:

  • Combine MHC Class II staining with cytokine detection to correlate expression with functional outcomes

  • Include markers for T cell activation when studying APC-T cell interactions

• Advanced application - dual MHC Class I and II detection:

  • When studying both MHC Class I and II expression, use spectrally distinct fluorochromes

  • Consider using MHC Class I (β2m) and Class II (Cd74) reporter mice for simultaneous visualization

What considerations should be made when storing and handling FITC-conjugated MHC Class II antibodies?

Proper storage and handling of FITC-conjugated MHC Class II antibodies is critical for maintaining optimal performance:

• Storage conditions:

  • Store at 2-8°C protected from prolonged exposure to light

  • Do not freeze the conjugated antibody

  • The typical formulation includes PBS with preservatives (e.g., 0.05% ProClin300, 1% BSA)

• Handling precautions:

  • ProClin is a POISONOUS AND HAZARDOUS SUBSTANCE that should be handled by trained staff only

  • Minimize repeated freeze-thaw cycles

  • Protect from light during all handling steps to prevent photobleaching

• Aliquoting recommendations:

  • For frequent use, consider creating small working aliquots to prevent repeated freeze-thaw cycles

  • Use amber tubes or wrap in aluminum foil to protect from light

• Shelf life considerations:

  • Check manufacturer's expiration date

  • Monitor performance over time using consistent positive controls

How can FITC-conjugated MHC Class II antibodies be used to study differential immune responses in disease models?

FITC-conjugated MHC Class II antibodies provide valuable tools for investigating immune dysregulation in disease states:

• Autoimmune disease research:

  • Track changes in MHC Class II expression levels on APCs in models of MS, rheumatoid arthritis, or lupus

  • Correlate MHC Class II expression with disease progression or therapeutic response

  • Investigate how differential expression of MHC Class II molecules influences T cell polarization toward Th1 or Th2 responses

• Inflammatory demyelination studies:

  • Use MHC Class II reporter mice combined with antibody validation to define dynamics in response to inflammatory demyelination

  • Investigate non-conventional antigen-presenting cells (e.g., oligodendroglia) that upregulate MHC Class II components during inflammation

• Haplotype-specific immune responses:

  • Compare MHC Class II expression levels between protective/suppressive haplotypes and disease-associated haplotypes

  • Correlate expression levels with cytokine production profiles and T cell differentiation patterns

• Experimental approach:

  • Use flow cytometry to quantify MHC Class II expression on specific cell populations

  • Combine with intracellular cytokine staining to correlate expression with functional outcomes

  • Apply immunofluorescence to visualize spatial distribution of MHC Class II+ cells in tissue sections

What controls are essential when using FITC-conjugated MHC Class II antibodies in experimental protocols?

Implementing appropriate controls is critical for accurate interpretation of results:

• Isotype control:

  • Use FITC-conjugated Rat IgG2b, κ isotype control at the same concentration as the primary antibody

  • Essential for determining background fluorescence and setting appropriate gates

• Haplotype controls:

  • Include both positive control samples (from H-2b, H-2d, H-2q, H-2u haplotype mice)

  • Include negative control samples (from H-2s or H-2f haplotype mice) to confirm specificity

• Biological controls:

  • Positive biological controls: LPS-activated B cells (known to show high MHC Class II expression)

  • Negative biological controls: Cell types not expected to express MHC Class II

• Technical controls:

  • Single-color controls for compensation when performing multi-color flow cytometry

  • Unstained controls to assess autofluorescence

  • Fluorescence-minus-one (FMO) controls to properly set gates in multi-parameter experiments

• Validation controls:

  • When using reporter systems, validate correlation between antibody staining and reporter signal using immunofluorescence or flow cytometry

Proper implementation of these controls ensures reliable and reproducible results in experiments utilizing FITC-conjugated MHC Class II antibodies.