CD90 Thy-1.2 Antibody

What is CD90.2 (Thy-1.2) and how does it differ from CD90.1?

CD90.2 (Thy-1.2) is an 18 kDa GPI-linked membrane glycoprotein belonging to the immunoglobulin superfamily. It functions primarily in cell adhesion regulation and signal transduction in T cells. The key distinction between CD90.2 and CD90.1 lies in their strain-specific expression patterns - CD90.2 is expressed in mouse strains including BALB/c, CBA, C3H, C57BL/6, C58/J, and SJL, while CD90.1 is expressed in PL and AKR strains. This allelic difference creates a critical experimental consideration when designing studies, as antibodies specific to CD90.2 (like clones 53-2.1 and 30-H12) will not recognize CD90.1-expressing cells .

The molecular variation between these alleles involves minimal amino acid differences that nevertheless create distinct epitopes recognized by separate monoclonal antibodies. This polymorphism is particularly valuable in adoptive transfer experiments where cells from CD90.1 and CD90.2 mice can be differentially tracked within the same recipient animal.

Which cell types express CD90.2 (Thy-1.2) and what is its functional significance?

CD90.2 demonstrates a complex expression pattern across multiple cell lineages. It is abundantly expressed on thymocytes and peripheral T lymphocytes in CD90.2-positive mouse strains, making it a reliable T cell marker in these models. Beyond lymphoid cells, CD90.2 is also expressed on neurons, neural cells, Kupffer's cells, fibroblasts, and various stromal cell populations .

Functionally, CD90.2 participates in several critical cellular processes:

• T cell activation and signal transduction

• Cell-cell adhesion and interaction

• Synaptogenesis in the central nervous system

• Interaction with CD45 in lymphocyte signaling cascades

• Regulation of hematopoietic stem cell differentiation

Its diverse expression pattern and involvement in multiple cellular processes make CD90.2 a valuable target for immunological, neurological, and stem cell research applications. The 30-H12 antibody has been documented to induce calcium flux in thymocytes and, when combined with anti-CD3/TCR antibodies, can promote thymocyte apoptosis while inhibiting proliferative responses in mature T cells .

How can I determine whether my experimental mouse strain expresses CD90.1 or CD90.2?

Determining the CD90 allelic variant in your experimental strain is essential before designing experiments using CD90.2 antibodies. The most reliable approach combines genotypic verification with phenotypic confirmation:

Methodological Approach:

• Literature verification: First, consult published strain information for common laboratory strains. BALB/c, CBA, C3H, C57BL/6, C58/J, and SJL are CD90.2-positive, while PL and AKR express CD90.1 .

• Flow cytometric validation: Isolate peripheral blood lymphocytes or splenocytes from your experimental strain and stain with fluorochrome-conjugated antibodies specific to CD90.1 and CD90.2. Include appropriate positive control samples from known CD90.1 and CD90.2 strains.

• PCR-based genotyping: For definitive determination, particularly in mixed backgrounds or newly developed strains, PCR-based genotyping targeting the single nucleotide polymorphisms distinguishing CD90.1 and CD90.2 provides conclusive verification.

This multi-faceted validation approach prevents experimental errors that might arise from incorrect assumptions about CD90 allelic expression in your model system.

What are the key differences between available CD90.2 antibody clones?

Several monoclonal antibody clones recognizing mouse CD90.2 are available, with 53-2.1 and 30-H12 being the most widely utilized. Understanding their distinct properties is crucial for selecting the appropriate reagent for specific applications:

The 53-2.1 clone has been extensively validated for flow cytometric analysis of mouse splenocytes at concentrations ≤0.06 μg per test (defined as antibody amount needed to stain a cell sample in 100 μL final volume) . The 30-H12 clone requires slightly higher concentrations (≤0.125 μg per test) for optimal staining .

For depletion experiments, the 30-H12 clone is preferred, particularly when using functional grade preparations. This clone has demonstrated efficacy in inducing calcium flux in thymocytes and, when combined with anti-CD3/TCR antibodies, promoting thymocyte apoptosis .

How should I optimize CD90.2 antibody concentration for flow cytometry applications?

Optimal antibody titration is essential for achieving high signal-to-noise ratio in flow cytometry experiments while minimizing non-specific binding and reagent waste. For CD90.2 antibodies, the following methodological approach is recommended:

Titration Protocol:

• Prepare single-cell suspensions from spleen or thymus of a CD90.2-positive mouse strain (e.g., C57BL/6).

• Adjust cell concentration to 1×10⁶ cells per sample.

• Create a titration series with 2-fold dilutions of the antibody starting from 0.5 μg and ending at approximately 0.008 μg per test (100 μL final volume).

• Include appropriate isotype control antibodies at equivalent concentrations.

• Analyze by flow cytometry, calculating the stain index for each concentration:
  Stain Index = (Median Positive - Median Negative) / (2 × Standard Deviation of Negative)

• Select the concentration providing the highest stain index while maintaining low background staining.

What are effective protocols for CD90.2-based cell isolation or depletion?

CD90.2 antibodies can be utilized for both positive selection of T cells and depletion of T cell populations in experimental settings:

Positive Magnetic Selection Protocol:

• Prepare single-cell suspension from spleen or lymph nodes in cold buffer (PBS + 0.5% BSA + 2mM EDTA).

• Count cells and incubate with biotin-conjugated anti-CD90.2 (clone 53-2.1) at 0.5 μg per 10⁷ cells for 20 minutes at 4°C.

• Wash cells and incubate with streptavidin-conjugated magnetic beads for 15 minutes at 4°C.

• Apply cell suspension to a magnetic separation column and collect both positive and negative fractions.

• Verify purity by flow cytometry using a different CD90.2 antibody clone or additional T cell markers.

In vivo T Cell Depletion Protocol:

• Select functional grade purified anti-CD90.2 antibody (preferably clone 30-H12) .

• Administer 250-500 μg of antibody intraperitoneally per mouse in 200 μL sterile PBS.

• For extended depletion, repeat injections every 3-4 days.

• Monitor depletion efficiency by analyzing blood samples using flow cytometry with alternative T cell markers.

• Include appropriate isotype control antibody injections in control animals.

The 30-H12 clone has demonstrated efficacy in depletion experiments due to its ability to induce calcium flux in thymocytes and promote thymocyte apoptosis when combined with anti-CD3/TCR antibodies . For depletion protocols, functional grade preparations free of sodium azide and low endotoxin levels are essential.

How can CD90.2 antibodies be effectively utilized in multiparameter flow cytometry?

Integrating CD90.2 antibodies into multiparameter flow cytometry panels requires careful consideration of fluorochrome selection, compensation, and antibody combinations to maximize information yield while minimizing spectral overlap:

Methodological Considerations:

• Fluorochrome selection: For high-expression targets like CD90.2 on T cells, lower brightness fluorochromes (e.g., FITC, PE) are sufficient. Reserve brighter fluorochromes (PE-Cy7, APC) for lower-expression antigens.

• Panel design optimization:

  • When using PE-conjugated anti-CD90.2 (clone 53-2.1), the optimal concentration is ≤0.03 μg per test .

  • For APC-conjugated anti-CD90.2, concentrations ≤0.06 μg per test are recommended .

  • Both conjugates are optimally excited by standard flow cytometer lasers (488-561 nm for PE, 633-647 nm for APC).

• Avoiding antibody interference: When analyzing multiple T cell markers, ensure epitope blocking is not occurring between antibodies targeting physically proximal epitopes.

• Sample preparation protocol:

  • Isolate lymphocytes from relevant tissues (spleen, thymus, lymph nodes)

  • Prepare single-cell suspensions (1×10⁶ cells per sample)

  • Block Fc receptors with anti-CD16/CD32 (10 minutes, 4°C)

  • Add optimally titrated fluorochrome-conjugated antibodies

  • Incubate 20-30 minutes at 4°C in the dark

  • Wash twice with flow buffer (PBS + 2% FBS + 0.1% sodium azide)

  • Analyze within 4 hours or fix with 1% paraformaldehyde

This approach enables precise identification and quantification of T cell subpopulations in complex immunological experimental settings.

What strategies can address epitope masking when using CD90.2 antibodies in combination with other T cell markers?

Epitope masking can occur when multiple antibodies targeting physically proximal epitopes are used simultaneously, resulting in competitive binding and false negative results. For CD90.2-targeted experiments, the following strategies can mitigate this issue:

Methodological Solutions:

• Sequential staining approach: First incubate with the antibody most prone to epitope masking, followed by washing and subsequent staining with additional markers. For CD90.2, this sequential approach is particularly important when combining with antibodies against molecules that interact with CD90, such as CD45 .

• Clone selection strategy: Utilize antibody clones known to recognize non-overlapping epitopes. For CD90.2, the 53-2.1 and 30-H12 clones recognize distinct epitope regions, making them potentially compatible in the same panel .

• Validation protocol:

  • Prepare three staining tubes with identical cell samples

  • Tube 1: Single-stain with anti-CD90.2 only

  • Tube 2: Single-stain with the potentially competing antibody

  • Tube 3: Combined staining with both antibodies

  • Compare fluorescence intensity of CD90.2 staining between tubes 1 and 3

  • A significant reduction in signal intensity in tube 3 indicates epitope masking

• Alternative marker strategy: If epitope masking cannot be resolved, consider using alternative T cell markers (CD3, TCRβ) in combination with CD90.2 to ensure comprehensive phenotyping.

By systematically addressing potential epitope masking, researchers can ensure accurate identification and characterization of T cell populations in complex experimental systems.

How can CD90.2 antibodies be employed in lineage tracing experiments?

CD90.2 provides an excellent marker for tracking T cell lineages in adoptive transfer experiments due to its stable expression and allelic variation. The following protocol outlines a methodological approach for CD90-based lineage tracing:

Adoptive Transfer Protocol with CD90 Allelic Differentiation:

• Donor cell preparation:

  • Isolate T cells from CD90.1 (congenic) donor mice using negative selection

  • Label a portion of cells with cell tracking dye (e.g., CFSE or CellTrace Violet) if proliferation tracking is desired

  • Verify purity and viability (>95% pure, >90% viable)

• Recipient preparation:

  • Select CD90.2-expressing recipient mice (e.g., C57BL/6)

  • If required, precondition recipients with sublethal irradiation (4-6 Gy)

• Transfer procedure:

  • Inject 1-5×10⁶ purified donor T cells intravenously into recipients

  • For tissue-specific homing studies, alternative routes may be employed

• Analysis strategy:

  • Harvest relevant tissues at experimental timepoints

  • Prepare single-cell suspensions

  • Stain with fluorochrome-conjugated antibodies against:

    • CD90.1 (to identify donor cells)

    • CD90.2 (to identify recipient cells)

    • Additional phenotypic markers of interest

  • Analyze by flow cytometry, distinguishing donor (CD90.1+) from recipient (CD90.2+) T cells

This approach leverages the allelic difference between CD90.1 and CD90.2 to create a powerful experimental system for tracking T cell migration, proliferation, and differentiation without requiring artificial labeling methods that might alter cellular function.

How should researchers validate CD90.2 antibody specificity and performance?

Thorough validation of CD90.2 antibodies is essential for generating reliable and reproducible experimental data. The following systematic approach ensures proper antibody performance:

Comprehensive Validation Strategy:

• Positive and negative control samples:

  • Positive controls: Thymocytes or peripheral T cells from CD90.2+ strains (C57BL/6, BALB/c)

  • Negative controls: Cells from CD90.1+ strains (AKR, PL) and non-T cell populations

  • The staining pattern should show clear separation between positive and negative populations

• Antibody titration:

  • Perform systematic titration as described previously

  • Document optimal concentration for specific applications

  • For clone 53-2.1, optimal concentrations are typically ≤0.06 μg per test

  • For clone 30-H12, optimal concentrations are typically ≤0.125 μg per test

• Lot-to-lot consistency testing:

  • When receiving a new antibody lot, compare staining patterns with previous lots

  • Calculate percent positive cells and mean fluorescence intensity

  • Acceptable variation should not exceed 10-15%

• Application-specific validation:

  • For flow cytometry: Verify specificity using fluorescence-minus-one (FMO) controls

  • For immunohistochemistry: Include isotype controls and blocking peptide controls

  • For depletion experiments: Confirm functional activity in pilot studies

Implementing this validation framework ensures reliable antibody performance across experimental applications and minimizes the risk of artifactual results due to reagent variability.

What are the most common technical issues when using CD90.2 antibodies and how can they be resolved?

Researchers commonly encounter several technical challenges when working with CD90.2 antibodies. Here are methodological solutions to address these issues:

Problem 1: High background staining in flow cytometry

• Causes: Fc receptor binding, non-specific binding, suboptimal antibody concentration

• Solutions:

  • Always block Fc receptors with anti-CD16/CD32 antibodies before staining

  • Increase washing steps (3× with excess buffer)

  • Optimize antibody concentration through proper titration

  • Include proper isotype controls matched for concentration and fluorochrome

Problem 2: Poor separation between positive and negative populations

• Causes: Suboptimal antibody clone, epitope masking, improper instrument settings

• Solutions:

  • Compare performance of different clones (53-2.1 vs. 30-H12)

  • Adjust PMT voltages to optimize separation

  • Consider alternative fluorochromes with higher stain index

  • Verify antibody performance using known positive controls

Problem 3: Inconsistent immunohistochemistry results with CD90.2 antibodies

• Causes: Suboptimal fixation, antigen masking, inefficient permeabilization

• Solutions:

  • Use freshly prepared 4% paraformaldehyde with short fixation times (10 minutes)

  • For frozen sections, acetone fixation often preserves CD90.2 epitopes better

  • Test multiple antigen retrieval methods, with emphasis on gentle methods

  • Both 53-2.1 and 30-H12 clones have been validated for immunohistochemical staining of frozen tissue sections

Problem 4: Inefficient depletion in vivo with anti-CD90.2 antibodies

• Causes: Insufficient antibody dose, inadequate administration schedule, neutralization

• Solutions:

  • Use functional grade preparations (free of sodium azide)

  • Increase antibody dose (250-500 μg per mouse)

  • Repeat injections every 3-4 days for maintained depletion

  • Clone 30-H12 is recommended for depletion experiments

Implementing these targeted troubleshooting approaches can significantly improve experimental outcomes when working with CD90.2 antibodies across various applications.

How do different tissue preparation methods affect CD90.2 epitope recognition?

CD90.2 epitope preservation and accessibility vary significantly across tissue preparation methods, affecting antibody binding and experimental outcomes:

Comparative Analysis of Preparation Methods:

Optimized Protocol for CD90.2 Detection in Frozen Sections:

• Collect tissue and snap-freeze in OCT compound using liquid nitrogen-cooled isopentane

• Section at 5-8 μm thickness and air-dry slides (1 hour minimum)

• Fix with ice-cold acetone for 10 minutes

• Air-dry sections completely (15-30 minutes)

• Rehydrate with PBS (3× washes)

• Block with 5% normal serum from the same species as the secondary antibody

• Apply primary anti-CD90.2 antibody (10 μg/mL) and incubate overnight at 4°C

• Wash extensively with PBS (3× for 5 minutes each)

• Apply appropriate detection system (fluorochrome-conjugated secondary antibody or streptavidin-biotin system)

• Counterstain and mount with appropriate medium

Both the 53-2.1 and 30-H12 clones have been validated for immunohistochemical staining of frozen tissue sections , with acetone fixation providing optimal epitope preservation while maintaining tissue morphology.

What emerging applications of CD90.2 antibodies should researchers consider?

CD90.2 antibodies continue to find utility in novel research applications beyond traditional T cell identification. Several emerging areas deserve consideration:

• Single-cell transcriptomics: CD90.2 antibodies conjugated to oligonucleotide barcodes are increasingly used in CITE-seq approaches, enabling simultaneous protein and transcript analysis at single-cell resolution.

• Engineered T cell therapies: CD90.2 can serve as a selection marker for murine CAR-T cells and other engineered T cell therapies in preclinical models, allowing tracking and isolation of modified cells.

• Neural stem cell research: Given CD90.2 expression on neural cells, these antibodies provide valuable tools for isolating and characterizing neural stem and progenitor populations from mouse models.

• Advanced imaging applications: Super-resolution microscopy techniques combined with fluorochrome-conjugated CD90.2 antibodies enable detailed analysis of T cell synapse formation and membrane dynamics.

These applications extend the utility of CD90.2 antibodies beyond conventional flow cytometry and immunohistochemistry, offering new avenues for integrated multi-omics approaches in immunology, neuroscience, and stem cell research.

What key considerations should guide experimental design when incorporating CD90.2 antibodies?

When designing experiments utilizing CD90.2 antibodies, researchers should consider several critical factors to ensure robust and interpretable results:

• Strain selection awareness: Always verify CD90 allelic expression (CD90.1 vs. CD90.2) in your experimental mouse strains before designing studies. This prevents false negative results when using strain-specific antibodies .

• Application-specific optimization: Different applications require distinct optimization approaches:

  • For flow cytometry: Optimize antibody concentration, fluorochrome selection, and compensation

  • For immunohistochemistry: Focus on fixation method and antigen retrieval

  • For depletion experiments: Consider dose, administration route, and kinetics

• Control implementation:

  • Include proper positive controls (T cells from CD90.2+ strains)

  • Use appropriate negative controls (non-T cells, cells from CD90.1+ strains)

  • Incorporate isotype controls at equivalent concentrations

• Alternative marker validation: Confirm key findings with additional T cell markers (CD3, TCR) to ensure observations are not artifacts of CD90.2 antibody binding characteristics.

By systematically addressing these considerations in experimental design, researchers can maximize the utility of CD90.2 antibodies while minimizing technical pitfalls that might compromise data interpretation.