CD90 Thy-1 Antibody

What is CD90/Thy-1 and why is it an important research target?

CD90, also known as Thy-1, is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that belongs to the immunoglobulin superfamily. It has a molecular weight of approximately 25-35 kDa and contains sites of glycosylation. CD90 plays crucial roles in cell adhesion and signaling pathways, particularly in immune cells and neuronal tissues. Its importance as a research target stems from its differential expression across multiple cell types and its involvement in various biological processes including immune regulation, stem cell maintenance, and neuronal development. CD90 is predominantly expressed on T cells, neurons, and certain stem cell populations, making it a valuable marker for identifying and isolating these cell types in research settings. Additionally, CD90's expression on hematopoietic stem cells, particularly when co-expressed with CD34, helps identify pluripotent cell populations essential for hematopoiesis and tissue regeneration . The study of CD90 has contributed significantly to our understanding of the immunoglobulin superfamily, as it is the smallest member and provided the first biochemical characterization of vertebrate GPI anchors .

What cell types express CD90/Thy-1 and how does expression differ across species?

CD90/Thy-1 is expressed across multiple cell types, with some notable species-specific differences in expression patterns:

Human CD90/Thy-1 Expression:

• Thymocytes

• CD34+ prothymocytes

• Hematopoietic stem cells

• Neurons

• A small subset of human fetal liver cells

• Cord blood cells

• Bone marrow cells

• CD34+ immature cells

• A distinct subset of mature CD34- cells that are CD3+CD4+

Mouse CD90/Thy-1 Expression:

• T cells

• Thymocytes

• Neural cells

• Kupffer's cells

• Fibroblasts

Common Features Across Species:
CD90 serves as a marker for various stem cell populations across species, including mesenchymal stem cells and cancer stem cells. In neuronal tissues across species, CD90 is expressed on the axonal processes of mature neurons. This conservation of expression in neuronal tissues suggests important functional roles in neuronal development and synaptogenesis .

The differential expression patterns between species should be carefully considered when selecting CD90 antibodies for research, as some antibodies show species-specific reactivity while others cross-react across human, mouse, and rat CD90 .

What are the common applications for CD90/Thy-1 antibodies in research?

CD90/Thy-1 antibodies are utilized across multiple research applications, each providing distinct insights into biological processes:

CD90 antibodies are especially valuable in stem cell research, where they help identify and isolate stem cell populations from various tissues. In neuroscience research, these antibodies facilitate the study of neuronal development and function. For immunological studies, CD90 antibodies are instrumental in investigating T cell development, activation, and regulation .

How do I select the appropriate CD90/Thy-1 antibody clone for my experiment?

Selecting the appropriate CD90/Thy-1 antibody clone requires careful consideration of several experimental parameters:

Species Reactivity:
Match the antibody's target species with your experimental model. Common clones include:

• aTHy-1A1: Reacts with mouse, rat, and human CD90

• eBio5E10 (5E10): Specifically recognizes human CD90

• F15-42-1: Reacts with human and monkey CD90

• G7: Mouse-specific (often used with biotin conjugation)

Application Compatibility:
Verify that the selected clone has been validated for your specific application:

• For flow cytometry: Prefer directly conjugated antibodies (PE, FITC, etc.)

• For Western blotting: Select clones validated for denatured protein recognition

• For immunohistochemistry: Check compatibility with your fixation method (formaldehyde, methanol, etc.)

Epitope Consideration:
Different clones recognize different epitopes on CD90, which may be differentially accessible depending on:

• Protein conformation

• Glycosylation status

• Interaction with other proteins

Conjugation Requirements:
Select based on your detection system:

• Unconjugated: For flexible secondary antibody detection

• Fluorochrome-conjugated (PE, FITC): For direct detection in flow cytometry or IF

• Enzyme-conjugated (HRP): For colorimetric detection

• Biotin-conjugated: For amplification with streptavidin systems

For critical experiments, it's advisable to test multiple clones under your specific conditions to identify the optimal performer. Review citation records and user reviews when available to gauge real-world performance in similar experimental setups .

How can I optimize CD90/Thy-1 antibody staining for multi-parameter flow cytometry?

Optimizing CD90/Thy-1 antibody staining for multi-parameter flow cytometry requires careful consideration of several technical factors:

Panel Design Considerations:

• Fluorophore Selection: CD90 is typically highly expressed on positive cells, making it compatible with less bright fluorophores like FITC. Reserve brighter fluorophores (PE, APC) for markers with lower expression levels. The eBio5E10 clone conjugated to PE has excitation at 488-561 nm and emission at 578 nm, making it compatible with blue, green, and yellow-green lasers .

• Spectral Overlap: Minimize fluorescence spillover by selecting fluorophores with minimal spectral overlap. When using PE-conjugated CD90 antibodies, avoid PE-Texas Red in the same panel unless your instrument has advanced compensation capabilities.

• Titration: Even pre-titrated antibodies like eBio5E10 (recommended at 5 μL/0.25 μg per test for 10^5-10^8 cells) should be titrated in your specific experimental system to determine optimal signal-to-noise ratios .

Staining Protocol Optimization:

• Sample Preparation: Ensure single-cell suspensions with high viability (>90%) to reduce non-specific binding.

• Blocking Strategy: Include 5-10% serum from the same species as the secondary antibody to reduce non-specific binding.

• Staining Temperature: CD90 staining is typically performed at 4°C for 30 minutes, but room temperature incubation may be preferable if co-staining with antibodies that recognize conformation-sensitive epitopes.

• Buffer Selection: PBS with 0.5-2% BSA or FBS and 0.02-0.05% sodium azide helps maintain cell viability and reduces non-specific binding.

Controls and Validation:

• Fluorescence Minus One (FMO): Essential for accurate gating, especially in multicolor panels.

• Isotype Controls: Use matched isotype controls (IgG2a κ for aTHy-1A1) to assess non-specific binding .

• Known Positive and Negative Populations: Human erythroleukemia (HEL) cells serve as a reliable positive control for CD90 expression .

Troubleshooting Common Issues:

• High Background: Increase blocking, reduce antibody concentration, or add an Fc receptor blocking step.

• Weak Signal: Check cell viability, increase antibody concentration, or switch to a brighter fluorophore.

• Population Separation Issues: Consider alternative clones or fluorophores; some clones may perform better in specific applications or with certain cell types .

What methodological approaches can resolve challenges in detecting CD90/Thy-1 in fixed tissue sections?

Detecting CD90/Thy-1 in fixed tissue sections presents several challenges due to the molecule's GPI anchor and sensitivity to fixation. Here are methodological approaches to overcome these challenges:

Fixation Protocol Optimization:

• Fixative Selection:

  • For frozen sections: 2-4% paraformaldehyde (PFA) for 10-15 minutes preserves CD90 epitopes while maintaining tissue morphology

  • For paraffin embedding: Use 10% neutral buffered formalin with fixation time limited to 24 hours

  • Avoid methanol fixation which can disrupt GPI-anchored proteins

• Antigen Retrieval Strategies:

  • Heat-induced epitope retrieval (HIER): Citrate buffer (pH 6.0) at 95-98°C for 20 minutes

  • For paraffin sections: Proteinase K digestion (20 μg/mL for 10-15 minutes at 37°C) may enhance detection

  • Combination approach: HIER followed by short protease treatment can significantly improve staining intensity

Staining Protocol Enhancements:

• Signal Amplification Systems:

  • Biotin-streptavidin: Use biotinylated secondary antibodies with streptavidin-HRP or streptavidin-fluorophore conjugates

  • Tyramide signal amplification (TSA): Particularly effective for tissues with low CD90 expression

  • Three-layer detection: Primary antibody → biotinylated secondary → streptavidin-HRP/fluorophore

• Background Reduction:

  • Extended blocking: 1-2 hours with 5-10% serum from the same species as the secondary antibody

  • Addition of 0.1-0.3% Triton X-100 to reduce non-specific binding (note: may affect membrane proteins)

  • Avidin/biotin blocking for tissues with endogenous biotin (liver, kidney)

Clone Selection Considerations:
Different anti-CD90 clones perform differently in fixed tissues:

• The aTHy-1A1 clone has been validated for paraffin-embedded sections (IHCP)

• For dual immunofluorescence, select clones with different host species to avoid cross-reactivity

• For weakly expressed CD90, consider using concentrated antibody formulations with extended incubation times (overnight at 4°C)

Positive Control Strategies:

• Include known CD90-positive tissues (thymus, brain sections) as procedural controls

• For human tissues, tonsil sections provide excellent positive controls with distinct CD90 expression patterns

• Perform parallel staining with frozen and fixed sections to validate fixation-sensitive epitopes

How can I quantitatively analyze CD90/Thy-1 expression across different experimental conditions?

Quantitative analysis of CD90/Thy-1 expression requires reliable measurement approaches tailored to different experimental platforms. Here are methodological strategies for various techniques:

Flow Cytometry Quantification:

• Relative Quantification:

  • Percent positive cells: Determine the proportion of CD90+ cells in your population

  • Mean/Median Fluorescence Intensity (MFI): Measure the average CD90 expression level per cell

  • Comparative analysis: Calculate fold change in MFI relative to control conditions

• Absolute Quantification:

  • Antibody Binding Capacity (ABC): Use calibration beads with known antibody binding capacities

  • Molecules of Equivalent Soluble Fluorochrome (MESF): Standardize results across experiments and instruments

  • Quantitative approach: Convert fluorescence to actual number of CD90 molecules per cell

Western Blot Densitometry:

• Protocol Optimization:

  • Load equal total protein (verified by housekeeping proteins like β-actin or GAPDH)

  • Include a standard curve of recombinant CD90 protein

  • Use advanced normalization with total protein stains (REVERT, Ponceau S)

• Analysis Approach:

  • Software-based quantification: ImageJ, ImageLab, or similar platforms

  • Normalization strategy: Express CD90 signal relative to loading controls

  • Statistical validation: Analyze technical and biological replicates

Immunohistochemistry Quantification:

• Morphometric Analysis:

  • Area-based measurements: Calculate percentage of CD90+ tissue area

  • Cell counting: Determine number of CD90+ cells per field or tissue area

  • Intensity grading: Score staining as negative, weak, moderate, or strong

• Digital Pathology Approaches:

  • Whole slide imaging with automated analysis

  • Machine learning algorithms for pattern recognition

  • Multiplex analysis for co-expression with other markers

Real-time qPCR for THY1 Gene Expression:

• Transcript Quantification:

  • Relative quantification: 2^-ΔΔCt method with appropriate reference genes

  • Absolute quantification: Standard curve using plasmid standards

  • Consideration of splice variants and isoforms

Data Integration and Validation:

• Cross-platform validation: Confirm protein expression changes with gene expression data

• Multiparametric analysis: Correlate CD90 expression with functional outcomes

• Longitudinal analysis: Track expression changes over time or treatment duration

When comparing experimental conditions, statistical approaches should match the data structure, with appropriate tests for parametric (t-test, ANOVA) or non-parametric (Mann-Whitney, Kruskal-Wallis) analyses.

What are the most effective strategies for isolating pure CD90/Thy-1 positive cell populations?

Isolating highly pure CD90/Thy-1 positive cell populations requires strategic application of separation techniques optimized for your specific experimental needs:

Magnetic-Activated Cell Sorting (MACS):

• Direct Magnetic Labeling:

  • Use anti-CD90 antibodies directly conjugated to magnetic microbeads

  • Typical yield: 85-95% purity with 70-90% recovery of target cells

  • Advantages: High throughput, relatively gentle, economical for large samples

• Indirect Magnetic Labeling:

  • Primary anti-CD90 antibody followed by magnetic microbead-conjugated secondary antibody

  • Useful when direct conjugates are unavailable or for signal amplification

  • Enhanced sensitivity: Can detect CD90 low-expressing cells

• Sequential MACS Protocol:

  • Perform negative selection to deplete major contaminating populations

  • Follow with positive selection for CD90+ cells

  • Can achieve >98% purity with minimal cell activation

Fluorescence-Activated Cell Sorting (FACS):

• Single-Marker Sorting:

  • Use PE or FITC-conjugated anti-CD90 antibodies (e.g., eBio5E10-PE at 5 μL per test)

  • Typical yield: >98% purity with 50-80% recovery depending on stringency

  • Advantages: Highest purity, can select based on expression level (high/medium/low)

• Multiparameter Sorting:

  • Combine CD90 with additional markers for isolating specific subpopulations

  • For hematopoietic stem cells: CD90+CD34+ co-expression identifies cells with long-term culture capability

  • For mesenchymal stem cells: CD90+CD73+CD105+ triple-positive selection

• Index Sorting:

  • Record fluorescence parameters of each sorted cell

  • Correlate phenotype with subsequent functional or single-cell analysis

Density Gradient Centrifugation Pre-enrichment:

• Protocol Enhancement:

  • Use Ficoll-Paque or Percoll gradients to pre-enrich mononuclear cells

  • Reduces sorting/separation time and improves yield

  • Particularly useful for blood, bone marrow, or disaggregated tissue samples

Negative Selection Approaches:

• Depletion Strategy:

  • Remove CD90-negative cells using antibody cocktails against lineage markers

  • Advantages: Cells remain untouched by selection antibodies

  • Useful for sensitive functional assays where receptor engagement might alter function

Optimization Parameters:

• Buffer Selection: PBS with 0.5% BSA and 2 mM EDTA minimizes cell clumping

• Temperature Control: Perform all steps at 4°C to reduce antibody capping and internalization

• Viability Discrimination: Include viability dyes to exclude dead cells (7-AAD, DAPI, PI)

For highest purity requirements, consider a two-step approach: MACS pre-enrichment followed by FACS purification, which can achieve >99% purity while maintaining higher cell recovery than FACS alone.

How should I design experiments to study CD90/Thy-1 function in stem cell populations?

Designing experiments to study CD90/Thy-1 function in stem cell populations requires careful consideration of experimental approaches that address both phenotypic and functional aspects:

Experimental Design Framework:

• Expression Profiling Studies:

  • Single-cell RNA sequencing: Characterize CD90 expression heterogeneity within stem cell populations

  • Co-expression analysis: Identify correlation between CD90 and other stem cell markers

  • Developmental trajectory: Track CD90 expression changes during differentiation using time-course experiments

• Functional Characterization:

  • Colony-forming assays: Compare colony-forming efficiency between CD90+ and CD90- populations

  • Differentiation potential: Assess multi-lineage differentiation capacity of CD90+ cells

  • Long-term culture assays: The CD90+CD34+ cell population shows enhanced capacity for long-term culture, suggesting a role in maintaining stemness

• Mechanistic Studies:

  • Knockdown/knockout approaches: Use siRNA, shRNA, or CRISPR-Cas9 to modulate CD90 expression

  • Overexpression systems: Express CD90 in CD90- cells to determine if stem cell properties are conferred

  • Signaling pathway analysis: Investigate downstream pathways activated by CD90 stimulation

Isolation and Characterization Protocol:

• Isolation Strategy:

  • Flow cytometry-based sorting of CD90+ cells using PE-conjugated antibodies

  • Magnetic separation with anti-CD90 microbeads

  • Establish purity verification through post-sort analysis

• Experimental Controls:

  • Positive controls: Known CD90+ stem cell populations (e.g., bone marrow-derived MSCs)

  • Negative controls: Terminally differentiated cells lacking CD90 expression

  • Isotype controls: To establish specificity of CD90 antibody staining

• Functional Readouts:

  • Self-renewal: Serial passaging and secondary colony formation

  • Differentiation: Expression of lineage-specific markers upon differentiation induction

  • In vivo reconstitution: Transplantation studies to assess engraftment potential

Advanced Approaches:

• Spatial Analysis:

  • In situ hybridization: Localize CD90 mRNA in stem cell niches

  • Multiplexed imaging: Co-localize CD90 with niche factors and other stem cell markers

  • Laser capture microdissection: Isolate CD90+ cells from specific tissue microenvironments

• Single-Cell Analysis:

  • Single-cell sorting: Functional analysis of individual CD90+ cells

  • Clonal expansion studies: Determine heterogeneity in differentiation potential

  • Correlation with stemness genes: Link CD90 expression levels to pluripotency factors

• Interaction Studies:

  • Co-culture experiments: Assess CD90's role in stem cell-niche cell interactions

  • Adhesion assays: Quantify the contribution of CD90 to cell-matrix or cell-cell adhesion

  • Conditioned media experiments: Investigate paracrine effects of CD90+ cells

When designing these experiments, it's critical to consider species-specific differences in CD90 expression and function, as these may limit the translatability of findings across experimental models.

What are common pitfalls in CD90/Thy-1 antibody-based experiments and how can they be addressed?

Researchers commonly encounter several technical challenges when working with CD90/Thy-1 antibodies. Here are the major pitfalls and strategic solutions:

Epitope Masking Issues:

• Problem: CD90's extensive glycosylation can mask epitopes recognized by certain antibody clones.
  Solution:

  • Try multiple antibody clones targeting different epitopes

  • Consider mild deglycosylation treatments for certain applications

  • For fixed tissues, optimize antigen retrieval methods (citrate buffer at pH 6.0)

• Problem: Protein-protein interactions may obscure CD90 epitopes in certain cellular contexts.
  Solution:

  • Modify fixation protocols to preserve epitope accessibility

  • Test both native and denaturing conditions in immunoblotting

  • Consider membrane isolation protocols to enhance detection

Cross-Reactivity Concerns:

• Problem: Antibodies may cross-react with related proteins, particularly across species.
  Solution:

  • Validate specificity using knockout/knockdown controls

  • Use species-specific antibodies when available (e.g., eBio5E10 for human CD90)

  • Perform absorption controls with recombinant CD90 protein

• Problem: Secondary antibody cross-reactivity in multi-color staining.
  Solution:

  • Use directly conjugated primary antibodies

  • Employ sequential staining with blocking steps between antibodies

  • Select secondary antibodies with minimal cross-reactivity to other species

Signal-to-Noise Optimization:

• Problem: High background in immunohistochemistry or immunofluorescence.
  Solution:

  • Extend blocking steps (1-2 hours with 5-10% serum)

  • Add 0.1-0.3% Triton X-100 for intracellular staining

  • Use Fab fragments to block endogenous immunoglobulins

• Problem: Weak signal detection in samples with low CD90 expression.
  Solution:

  • Implement signal amplification systems (biotin-streptavidin, tyramide)

  • Increase antibody concentration or incubation time

  • Switch to more sensitive detection methods (ECL Prime for Western blots)

Functional Impact Concerns:

• Problem: Antibody binding may trigger signaling or alter cell behavior.
  Solution:

  • Use Fab fragments for functional studies

  • Include appropriate isotype controls in all experiments

  • Consider genetic approaches (siRNA, CRISPR) as complementary strategies

• Problem: Antibody internalization affecting long-term experiments.
  Solution:

  • Perform kinetic studies to determine internalization rate

  • Use fixation to "freeze" the staining at specific timepoints

  • Consider non-antibody based detection methods for live-cell tracking

Reproducibility Challenges:

• Problem: Lot-to-lot variability in antibody performance.
  Solution:

  • Validate each new lot against previous standards

  • Maintain internal positive controls across experiments

  • Consider pooling antibody lots for long-term studies

• Problem: Protocol drift between different operators or laboratories.
  Solution:

  • Develop detailed standard operating procedures (SOPs)

  • Implement quality control metrics for staining intensity

  • Use automated systems where possible to reduce variability

How do CD90/Thy-1 expression patterns change in disease models and what are the best methods to quantify these changes?

CD90/Thy-1 expression undergoes significant alterations in various disease contexts, requiring specialized methodological approaches for accurate quantification:

Cancer Models:

• Expression Pattern Changes:

  • Upregulation in cancer stem cells across multiple tumor types

  • Association with epithelial-to-mesenchymal transition (EMT)

  • Correlation with invasion, metastasis, and treatment resistance

  • Implication in nasopharyngeal carcinoma and thymoma pathogenesis

• Quantification Methods:

  • Flow cytometry: Determine percentage of CD90+ cells and expression intensity in tumor samples

  • Immunohistochemistry: Assess CD90 distribution within tumor microenvironment using tissue microarrays

  • Single-cell RNA-seq: Characterize heterogeneity of CD90 expression in tumor cell subpopulations

  • Circulating tumor cell analysis: Evaluate CD90+ CTCs as potential biomarkers

Fibrotic Diseases:

• Expression Pattern Changes:

  • Increased CD90 expression in activated myofibroblasts

  • Altered distribution in fibrotic tissues versus healthy controls

  • Dynamic changes during disease progression

• Quantification Methods:

  • Dual immunofluorescence: Co-localize CD90 with other fibrosis markers (α-SMA, collagen)

  • Digital pathology: Quantify fibrotic area with CD90 positivity

  • Western blotting: Compare CD90 protein levels in fibrotic versus normal tissues

  • qRT-PCR: Measure THY1 mRNA upregulation during fibrogenesis

Autoimmune Disorders:

• Expression Pattern Changes:

  • Altered CD90 expression on T lymphocytes

  • Association with disease activity in models of graft-versus-host disease

  • Changes in CD90+ regulatory T cell populations

• Quantification Methods:

  • Multi-parameter flow cytometry: Characterize CD90+ T cell subsets

  • Cytokine production analysis: Correlate CD90 expression with functional cytokine profiles

  • In vivo imaging: Track CD90+ cell migration in animal models

  • Phospho-flow cytometry: Link CD90 expression to signaling pathway activation

Neurodegenerative Conditions:

• Expression Pattern Changes:

  • Modulation of CD90 expression on neurons in response to injury

  • Altered distribution in axonal processes during neurodegeneration

  • Changes in CD90+ neural stem/progenitor cells

• Quantification Methods:

  • Laser scanning cytometry: Quantify CD90 expression in tissue sections

  • 3D confocal microscopy: Analyze spatial distribution of CD90 in neural tissues

  • Electrophysiological correlation: Link CD90 expression to neuronal function

  • Brain organoid models: Track CD90 expression during development and disease progression

Statistical Analysis Approaches:

• Categorical Analysis:

  • Chi-square tests for positive/negative classification

  • Scoring systems (0, 1+, 2+, 3+) with weighted kappa statistics for inter-observer agreement

  • Receiver operating characteristic (ROC) analysis to determine optimal cutpoints

• Continuous Variable Analysis:

  • Mann-Whitney or t-tests for two-group comparisons

  • ANOVA with post-hoc testing for multiple group comparisons

  • Correlation analysis between CD90 expression and clinical parameters

  • Survival analysis (Kaplan-Meier, Cox regression) for prognostic value assessment

The selection of quantification method should align with the specific disease model and research question, with consideration for both technical feasibility and biological relevance.

How can I effectively use CD90/Thy-1 antibodies in multiplex immunofluorescence or mass cytometry experiments?

Integrating CD90/Thy-1 antibodies into multiplex immunofluorescence or mass cytometry experiments requires strategic panel design and technical optimization to achieve reliable and informative results:

Multiplex Immunofluorescence Optimization:

• Panel Design Considerations:

  • Fluorophore Selection: For CD90, which is typically highly expressed, moderate brightness fluorophores like FITC are often sufficient. Reserve brighter fluorophores (Alexa Fluor 647, PE) for lower-expressed targets.

  • Spectral Compatibility: Design panels minimizing spectral overlap; when using CD90-PE (emission ~578 nm), avoid PE-Texas Red unless using spectral unmixing.

  • Antibody Pairing: When combining with other markers, confirm antibodies are raised in different host species or use directly conjugated antibodies to prevent cross-reactivity.

• Sequential Staining Protocols:

  • Tyramide Signal Amplification (TSA): For tissues with low CD90 expression, implement TSA-based multiplex protocols:

    1. Stain with anti-CD90 primary antibody

    2. Apply HRP-conjugated secondary antibody

    3. Develop with tyramide-fluorophore

    4. Heat-inactivate HRP

    5. Proceed to next marker

  • Stripping and Reprobing: For non-TSA approaches, antibody stripping with glycine-SDS buffer (pH 2.0) followed by re-staining can enable sequential detection of multiple markers.

• Spectral Unmixing Approaches:

  • Implement linear unmixing algorithms for overlapping fluorophores

  • Include single-stained controls for each fluorophore

  • Utilize autofluorescence subtraction for tissues with high background (brain, liver)

Mass Cytometry (CyTOF) Implementation:

• Metal Isotope Selection:

  • Choose isotope abundance based on CD90 expression level:

    • For high expression: Lower abundance isotopes may be sufficient

    • For variable expression: Higher abundance isotopes provide better resolution

  • Avoid isotopes with known oxide formation or spillover issues

• Antibody Conjugation Strategy:

  • Commercial pre-conjugated anti-CD90 antibodies

  • Custom conjugation using MaxPAR or other metal-labeling kits

  • Validate metal-conjugated antibodies against fluorochrome-conjugated versions

• Panel Development:

  • Include CD90 in stem cell or immunophenotyping panels

  • Combine with lineage markers, activation markers, and functional readouts

  • Design panels according to experimental questions (e.g., stem cell identification, immune profiling)

Validation and Quality Control:

• Control Samples:

  • Single-stained controls: Essential for compensation/unmixing

  • FMO controls: Critical for accurate gating, especially with numerous parameters

  • Biological controls: Include known CD90+ and CD90- populations

• Antibody Validation:

  • Titrate each antibody individually before multiplexing

  • Confirm staining pattern matches expected distribution

  • Validate in relevant tissues/cells (e.g., thymus, neurons, stem cells)

• Data Analysis Approaches:

  • Dimension Reduction: tSNE, UMAP for visualization of high-parameter data

  • Clustering Algorithms: FlowSOM, PhenoGraph for population identification

  • Trajectory Analysis: Diffusion maps, pseudotime for developmental studies

Practical Protocol Example:

For a 7-color multiplex immunofluorescence panel including CD90:

• Sample Preparation:

  • Fix tissue samples in 4% PFA for 24 hours

  • Process to paraffin or prepare frozen sections

  • For FFPE tissues, perform antigen retrieval in citrate buffer (pH 6.0)

• Blocking:

  • 10% normal goat serum in PBS with 0.3% Triton X-100 for 2 hours

  • Additional avidin/biotin blocking if using biotin-based detection

• Primary Antibody Application:

  • Anti-CD90 antibody (aTHy-1A1 or eBio5E10) at optimized concentration

  • Incubate overnight at 4°C in humidity chamber

  • Include markers of interest (lineage, activation, functional markers)

• Detection System:

  • Directly conjugated antibodies: Wash and proceed to next marker

  • TSA-based detection: Apply appropriate HRP-secondary, develop with tyramide-fluorophore, quench with H₂O₂

  • Apply DAPI for nuclear counterstain

• Image Acquisition:

  • Multispectral imaging with appropriate filter sets

  • Capture regions of interest and representative areas

  • Include single-stained controls on same slide/session

This approach enables comprehensive characterization of CD90+ cells in relation to their microenvironment, functional status, and developmental stage.

What is the significance of CD90/Thy-1 as a marker in stem cell research and regenerative medicine?

CD90/Thy-1 has emerged as a critical marker in stem cell biology with significant implications for regenerative medicine applications:

Stem Cell Identification and Isolation:

• Mesenchymal Stem Cells (MSCs):

  • CD90 serves as one of the three defining markers for MSCs (along with CD73 and CD105)

  • High, stable expression throughout passages makes CD90 a reliable MSC identifier

  • Used in quality control for clinical-grade MSC production

  • Enables prospective isolation of MSCs from heterogeneous tissue digests

• Hematopoietic Stem Cells (HSCs):

  • CD90+CD34+ population is enriched for cells with long-term culture capabilities

  • CD90 expression distinguishes primitive HSCs from more committed progenitors

  • Expression levels correlate with self-renewal versus differentiation potential

  • Useful for monitoring HSC mobilization and engraftment in transplantation settings

• Tissue-Resident Stem Cells:

  • CD90 marks stem/progenitor populations in multiple tissues:

    • Liver progenitor cells (oval cells)

    • Dental pulp stem cells

    • Cardiac progenitors

    • Limbal stem cells of the cornea

Functional Relevance in Regenerative Processes:

• Differentiation Regulation:

  • CD90 modulation accompanies lineage commitment in multiple stem cell types

  • Expression changes correlate with acquisition of specialized functions

  • May function as a checkpoint in differentiation pathways

• Immunomodulatory Properties:

  • CD90+ MSCs display enhanced immunosuppressive capacities

  • Critical for therapeutic applications in inflammatory and autoimmune conditions

  • Mediates interactions with immune cells to create favorable regenerative environments

• Tissue Regeneration Dynamics:

  • Upregulation in response to tissue injury

  • Involvement in wound healing and tissue remodeling

  • Potential target for enhancing endogenous repair mechanisms

Translational Applications:

• Cell Therapy Quality Control:

  • Standard release criterion for clinical-grade MSCs

  • Flow cytometric assessment ensures >95% CD90 positivity

  • Used to track cell product stability during manufacturing and storage

• Biomarker Applications:

  • Circulating CD90+ cells as indicators of regenerative capacity

  • Tissue expression patterns as predictors of healing potential

  • Response biomarker for regenerative interventions

• Therapeutic Target Development:

  • CD90-targeting strategies to mobilize endogenous stem cells

  • Modulation of CD90 signaling to enhance regenerative functions

  • Engineered CD90 expression to improve cellular therapeutics

Emerging Research Directions:

• Single-Cell Technologies:

  • Heterogeneity within CD90+ populations

  • Correlation with functional stem cell properties at single-cell resolution

  • Identification of CD90+ subpopulations with enhanced regenerative potential

• Biomaterial Integration:

  • CD90-binding scaffolds for stem cell delivery

  • Surface modifications to enhance CD90+ cell retention

  • Controlled release systems targeting CD90-mediated pathways

• In Vivo Tracking:

  • Non-invasive monitoring of CD90+ cells after transplantation

  • Correlation of CD90+ cell persistence with therapeutic outcomes

  • Real-time assessment of stem cell engraftment and differentiation

The significance of CD90 continues to expand as our understanding of its functional roles in stem cell biology deepens, with implications for both basic science and clinical translation in regenerative medicine.

How do different CD90/Thy-1 epitopes correlate with functional outcomes in research applications?

The relationship between CD90/Thy-1 epitopes and functional outcomes is a critical consideration in research design, as different epitopes can reveal distinct biological aspects of this multifunctional molecule:

Structural Epitope Mapping and Functional Correlations:

• N-Terminal Domain Epitopes:

  • Recognized by: eBio5E10 (5E10) clone

  • Functional Correlation: These epitopes are associated with T cell activation and signal transduction

  • Research Application: Ideal for studying CD90's role in immunological synapse formation

  • Epitope Stability: Generally resistant to fixation, suitable for FFPE tissue analysis

• GPI Anchor-Proximal Epitopes:

  • Recognized by: Specific monoclonal antibodies targeting membrane-proximal regions

  • Functional Correlation: These epitopes relate to lipid raft association and membrane organization

  • Research Application: Valuable for investigating CD90's role in organizing signaling complexes

  • Epitope Sensitivity: Often disrupted by harsh detergents or fixatives, requiring gentle processing

• Glycosylation-Dependent Epitopes:

  • Recognized by: Various clones with differential sensitivity to glycosylation status

  • Functional Correlation: Glycosylation patterns reflect cell state and differentiation status

  • Research Application: Useful for discriminating stem cell subpopulations with different potency

  • Epitope Variability: May show heterogeneity based on cell type or activation state

Epitope-Specific Experimental Outcomes:

• Stem Cell Research Applications:

  • Observation: Antibodies recognizing specific glycosylated epitopes identify stem cells with enhanced pluripotency

  • Functional Outcome: Isolation of cells with superior colony-forming efficiency and differentiation potential

  • Methodological Implication: Selection of appropriate clones critically affects stem cell yield and quality

• Neuronal Function Studies:

  • Observation: Antibodies binding to neuronal-specific CD90 epitopes affect neurite outgrowth

  • Functional Outcome: Different epitope binding can either promote or inhibit neuronal differentiation

  • Methodological Implication: Epitope selection influences outcomes in neural regeneration experiments

• Immunological Research:

  • Observation: Certain epitope-specific antibodies trigger signaling cascades while others are functionally neutral

  • Functional Outcome: Signaling-inducing antibodies may confound results in functional assays

  • Methodological Implication: For pathway analysis, non-activating epitope binding is preferable

Clone-Specific Performance Characteristics:

Epitope Accessibility Considerations:

• Membrane Conformation Effects:

  • Certain epitopes are masked or revealed depending on CD90's association with other membrane proteins

  • Cell activation can alter epitope accessibility through conformational changes

  • Methodological solution: Try multiple clones when investigating novel cell types or conditions

• Fixation-Dependent Epitope Alterations:

  • Aldehyde fixation can mask certain CD90 epitopes while preserving others

  • Alcohol fixation often preserves different epitope sets than aldehydes

  • Methodological solution: Optimize fixation protocols for specific applications and clones

• Protein-Protein Interaction Interference:

  • Some epitopes overlap with binding sites for natural CD90 ligands

  • Antibody binding may compete with or mimic natural interactions

  • Methodological solution: Compare results using antibodies targeting non-overlapping epitopes

Understanding these epitope-function relationships enables researchers to select appropriate antibody clones based on their specific experimental questions and anticipated functional outcomes.

What are the emerging research directions for CD90/Thy-1 antibodies in academic studies?

The scientific landscape surrounding CD90/Thy-1 antibodies is rapidly evolving, with several innovative research directions emerging at the intersection of technology advancement and biological discovery:

Advanced Imaging Applications:

• Super-Resolution Microscopy:

  • Nanoscopic visualization of CD90 distribution in membrane microdomains

  • Investigation of CD90's role in organizing signaling clusters at unprecedented resolution

  • Combined with proximity ligation assays to identify molecular interaction partners at nanoscale

• Intravital Imaging:

  • Real-time tracking of CD90+ cells in living organisms using fluorescent anti-CD90 antibodies

  • Monitoring stem cell migration, differentiation, and interaction with niche components

  • Correlation of spatial dynamics with functional outcomes in regenerative processes

• Correlative Light-Electron Microscopy:

  • Precise localization of CD90 at ultrastructural level

  • Investigation of CD90's relationship with subcellular structures

  • Resolving the nanoscale organization of CD90 in specialized membrane domains

Multi-Omics Integration:

• Antibody-Based Spatial Transcriptomics:

  • Using CD90 antibodies to anchor spatial transcriptomic analyses of stem cell niches

  • Correlation of CD90 protein expression with local transcriptional landscapes

  • Identification of spatially restricted gene expression programs in CD90+ cells

• Phospho-Proteomics:

  • Antibody-based isolation of CD90+ cells followed by phospho-proteomic analysis

  • Mapping signaling networks downstream of CD90 engagement

  • Comparison of phosphorylation landscapes in different CD90+ cell populations

• Single-Cell Multi-Parameter Analysis:

  • Integration of CD90 antibody staining with single-cell RNA-seq or ATAC-seq

  • Correlation of CD90 protein levels with transcriptional or epigenetic states

  • Identification of CD90-associated regulatory networks at single-cell resolution

Engineered Antibody Derivatives:

• Bispecific Antibodies:

  • CD90-targeting bispecific antibodies for selective immune cell recruitment

  • Dual targeting of CD90 and differentiation-inducing receptors

  • Combined targeting of CD90 with tissue-specific antigens for improved specificity

• Antibody-Drug Conjugates:

  • Selective delivery of therapeutics to CD90+ cells using antibody carriers

  • Application in elimination of pathological CD90+ populations (cancer stem cells)

  • Controlled modification of stem cell behavior through targeted payload delivery

• Nanobodies and Alternative Scaffolds:

  • Development of camelid nanobodies against CD90 for improved tissue penetration

  • Application in difficult-to-access tissues or intravital imaging

  • Enhanced multiplexing capabilities due to smaller size and different species origin

Functional Modulation Applications:

• Activating/Inhibitory Antibodies:

  • Development of antibodies that specifically activate or inhibit CD90 signaling

  • Application in directed differentiation protocols for stem cells

  • Therapeutic modulation of CD90+ cells in disease settings

• Conditional Depletion Strategies:

  • Anti-CD90 antibody-based ADC systems for selective cell ablation

  • Temporal control of CD90+ cell depletion in developmental studies

  • Investigation of CD90+ cell functions through acute elimination approaches

• Chimeric Antigen Receptor (CAR) Development:

  • CD90-directed CAR-T cells for targeting pathological stem cell populations

  • Fine-tuned recognition of CD90 epitopes for improved specificity

  • Application in resistant malignancies with CD90+ cancer stem cell populations

These emerging directions highlight the continuing evolution of CD90/Thy-1 antibodies from simple detection tools to sophisticated reagents enabling mechanistic insights and therapeutic applications across multiple fields of biomedical research.

How can researchers integrate CD90/Thy-1 antibodies with emerging single-cell and spatial biology technologies?

The integration of CD90/Thy-1 antibodies with cutting-edge single-cell and spatial biology technologies is creating unprecedented opportunities for understanding cellular heterogeneity and tissue architecture:

Single-Cell Technology Integration:

• CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing):

  • Methodological Approach: Use oligo-tagged anti-CD90 antibodies in conjunction with single-cell RNA sequencing

  • Implementation Strategy:

    1. Label cells with oligonucleotide-conjugated anti-CD90 antibodies

    2. Perform single-cell RNA capture and sequencing

    3. Simultaneously quantify CD90 protein expression and transcriptome profiles

  • Research Applications:

    • Correlation of CD90 protein levels with gene expression signatures

    • Identification of transcriptional states specific to CD90high versus CD90low populations

    • Discovery of novel CD90+ cell subpopulations with distinct functional properties

• Flow-Seq and Index Sorting:

  • Methodological Approach: Use flow cytometry with CD90 antibodies to isolate cells for downstream single-cell sequencing

  • Implementation Strategy:

    1. Stain cells with fluorochrome-conjugated anti-CD90 antibodies

    2. Sort single cells while recording (indexing) their fluorescence intensity

    3. Perform single-cell sequencing on sorted cells

  • Research Applications:

    • Precise correlation of CD90 expression level with genomic, transcriptomic, or epigenomic features

    • Selection of rare CD90+ subpopulations for detailed molecular characterization

    • Retrospective analysis of sequenced cells based on CD90 expression intensity

• Single-Cell Proteomics:

  • Methodological Approach: Incorporate anti-CD90 antibodies into mass cytometry or single-cell Western blot technologies

  • Implementation Strategy:

    1. Use metal-conjugated anti-CD90 antibodies for CyTOF analysis

    2. Integrate with dozens of other protein markers

    3. Analyze with high-dimensional clustering algorithms

  • Research Applications:

    • Mapping protein co-expression networks in CD90+ cells

    • Identifying signaling states correlated with CD90 expression

    • Characterizing post-translational modifications in CD90+ populations

Spatial Biology Applications:

• Multiplex Immunofluorescence and Imaging Mass Cytometry:

  • Methodological Approach: Combine anti-CD90 antibodies with spatial profiling technologies

  • Implementation Strategy:

    1. Apply anti-CD90 antibodies in multiplex panels (CODEX, MIBI, Hyperion)

    2. Image tissue sections with subcellular resolution

    3. Analyze spatial relationships between CD90+ cells and their microenvironment

  • Research Applications:

    • Mapping CD90+ cell distribution in complex tissues

    • Quantifying spatial relationships with other cell types

    • Identifying tissue niches supporting CD90+ cells

• Spatial Transcriptomics with Protein Integration:

  • Methodological Approach: Combine CD90 immunostaining with spatial transcriptomics technologies

  • Implementation Strategy:

    1. Perform CD90 immunofluorescence on tissue sections

    2. Apply spatial transcriptomics platform (10x Visium, Slide-seq, MERFISH)

    3. Register antibody staining with transcriptome data

  • Research Applications:

    • Defining transcriptional landscapes in and around CD90+ cell niches

    • Discovering spatial gene expression patterns correlated with CD90 gradients

    • Identifying ligand-receptor pairs involved in CD90+ cell communication

• In Situ Sequencing and Imaging:

  • Methodological Approach: Visualize CD90 protein together with targeted RNA profiles

  • Implementation Strategy:

    1. Combine anti-CD90 immunofluorescence with in situ RNA detection

    2. Apply padlock probes or FISH for key genes of interest

    3. Capture multichannel images for co-localization analysis

  • Research Applications:

    • Correlating CD90 protein expression with local gene expression

    • Validating transcriptional states in spatially resolved contexts

    • Investigating RNA-protein relationships at single-cell resolution

Integration Challenges and Solutions:

• Technical Compatibility Issues:

  • Challenge: Some fixation protocols for sequencing may compromise CD90 epitopes

  • Solution: Optimize bifunctional protocols that preserve both protein epitopes and nucleic acid integrity

  • Approach: Test cross-linking fixatives at reduced concentrations or implement adaptive fixation timeframes

• Data Integration Complexities:

  • Challenge: Aligning protein expression data with transcriptomic or genomic datasets

  • Solution: Develop computational pipelines specifically designed for multi-modal data integration

  • Approach: Apply machine learning algorithms to identify correlations between protein and RNA measurements

• Sensitivity Limitations:

  • Challenge: Different sensitivity thresholds between antibody detection and sequencing technologies

  • Solution: Implement signal amplification strategies for protein detection

  • Approach: Use tyramide signal amplification or photonic crystal enhancement to boost antibody signals