TNFRSF8 Mouse

What is mouse CD30/TNFRSF8 and how does it differ structurally from human CD30?

Mouse CD30 (TNFRSF8) is a 120 kDa type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily. The mature mouse CD30 protein consists of three distinct domains: a 264 amino acid extracellular domain (ECD) containing three cysteine-rich repeats, a 27 amino acid transmembrane segment, and a 190 amino acid cytoplasmic domain. Unlike mouse CD30, human CD30 contains an additional 90 amino acids in the ECD and features six cysteine-rich repeats instead of three. Within shared regions of the ECD, mouse CD30 exhibits 53% amino acid sequence identity with human CD30 and 80% with rat CD30 .

When designing cross-species experiments, these structural differences must be considered, particularly for antibody selection, as epitope recognition may vary significantly between species. Researchers should validate antibody cross-reactivity before conducting comparative studies.

What are the normal expression patterns of CD30/TNFRSF8 in mouse tissues?

Under physiological conditions, CD30/TNFRSF8 expression is predominantly restricted to activated T and B lymphocytes. It is not typically expressed on resting lymphocytes. Expression is induced following antigen stimulation of both Th cells and B cells . In experimental settings, CD30 expression can be detected in mouse splenocytes using immunofluorescence techniques with specific anti-CD30 antibodies .

For tissue-specific expression studies, researchers should consider using multiple detection methods including flow cytometry, immunohistochemistry, and mRNA analysis to comprehensively map expression patterns across different activation states and tissue microenvironments.

What signaling pathways does mouse CD30/TNFRSF8 activate and what are their functional outcomes?

Mouse CD30/TNFRSF8 primarily exerts its effects through engaging with its ligand, CD30L/TNFSF8, which is expressed on activated Th cells, monocytes, granulocytes, and medullary thymic epithelial cells. This interaction triggers several important functional outcomes:

• Costimulation of antigen-induced Th0 and Th2 proliferation and cytokine secretion

• Promotion of Th2-biased immune responses

• Induction of IL-13 expression in T cells, even in the absence of antigenic stimulation

• Contribution to thymic negative selection through apoptotic cell death of CD4+CD8+ T cells

For investigating these signaling pathways, researchers should employ techniques such as western blotting for downstream phosphorylation events, gene expression analysis for cytokine production, and cell viability assays to assess apoptotic outcomes.

What are the recommended methods for detecting mouse CD30/TNFRSF8 in experimental samples?

Several validated methodologies exist for detecting mouse CD30/TNFRSF8:

• ELISA: Sandwich ELISA using specific capture and detection antibodies allows quantification of soluble CD30 in serum, plasma, and cell culture media. The typical detection range for commercial ELISA kits is 19.5-1250 pg/mL .

• Immunofluorescence: Detection of CD30 on cellular surfaces can be performed using fluorochrome-conjugated antibodies. For example, CD30 has been successfully detected in immersion-fixed mouse splenocytes using goat anti-mouse CD30/TNFRSF8 antibodies followed by fluorescent secondary antibodies .

• Flow Cytometry: For quantitative analysis of CD30 expression on specific cell populations, flow cytometry using validated anti-CD30 antibodies is recommended.

When selecting a detection method, researchers should consider the specific experimental question, sample type, and required sensitivity. For low-abundance samples, more sensitive techniques such as ELISA may be preferable over immunohistochemistry.

How should mouse CD30/TNFRSF8 recombinant proteins and antibodies be stored and handled to maintain activity?

Proper storage and handling of CD30/TNFRSF8 research reagents is crucial for experimental success:

• Recombinant Proteins:

  • Store lyophilized proteins at -20°C to -80°C for up to 12 months

  • Reconstituted protein solutions can be stored at 4-8°C for 2-7 days

  • For longer storage, aliquot and maintain at < -20°C for up to 3 months

• Antibodies:

  • Use a manual defrost freezer and avoid repeated freeze-thaw cycles

  • Store at -20°C to -70°C for up to 12 months from receipt date (as supplied)

  • After reconstitution, store at 2-8°C under sterile conditions for up to 1 month

  • For longer storage after reconstitution, maintain at -20°C to -70°C under sterile conditions for up to 6 months

Researchers should always follow manufacturer-specific instructions as optimal storage conditions may vary between product formulations.

What controls should be included when measuring mouse CD30/TNFRSF8 in experimental systems?

Robust experimental design for CD30/TNFRSF8 analysis should include several types of controls:

• Positive Controls:

  • Activated mouse T cells known to express CD30

  • Cell lines transfected with mouse CD30

  • Recombinant mouse CD30 protein (for ELISA)

• Negative Controls:

  • Resting lymphocytes that do not express CD30

  • Samples from CD30 knockout mice

  • Isotype control antibodies to assess non-specific binding

• Technical Controls:

  • Standard curves using recombinant proteins of known concentration

  • Internal reference standards to normalize across experimental batches

  • Dilution linearity tests to confirm detection within the quantitative range

Each experimental method requires specific controls. For example, ELISA experiments should include blank wells and standard curves, while flow cytometry should include fluorescence-minus-one (FMO) controls and isotype controls.

How do mouse models of CD30/TNFRSF8 function compare to human systems in immunological research?

While mouse CD30/TNFRSF8 shares functional similarities with human CD30, several important differences must be considered in translational research:

• Structural Differences: Mouse CD30 contains three cysteine-rich repeats in its ECD compared to six in human CD30, and shares only 53% sequence identity in common regions . These differences may affect ligand binding properties and downstream signaling dynamics.

• Expression Patterns: Although both mouse and human CD30 are expressed on activated lymphocytes and upregulated in certain pathological conditions, tissue-specific and temporal expression patterns may differ.

• Signaling Outcomes: While both mouse and human CD30 promote Th2-biased responses, the magnitude and specific cytokine profiles induced may vary between species.

What experimental approaches can distinguish between membrane-bound and soluble forms of mouse CD30/TNFRSF8?

Distinguishing between membrane-bound and soluble CD30 requires specific methodological approaches:

• For Membrane-bound CD30:

  • Flow cytometry using anti-CD30 antibodies on intact cells

  • Immunofluorescence microscopy of non-permeabilized cells

  • Cell surface biotinylation followed by immunoprecipitation

• For Soluble CD30:

  • ELISA-based detection in cell-free supernatants, serum, or plasma

  • Western blotting of concentrated supernatants using antibodies against the extracellular domain

  • Size-exclusion chromatography to separate soluble CD30 from membrane-bound forms in vesicles

When studying the relationship between these forms, researchers can employ techniques such as inhibitors of metalloproteinases to block shedding, or stimulate shedding with phorbol esters to assess the dynamics of conversion from membrane-bound to soluble forms.

How can mouse CD30/TNFRSF8 signaling be manipulated in experimental systems to study its role in immune regulation?

Several experimental approaches can be used to manipulate CD30 signaling:

• Genetic Approaches:

  • CD30 knockout mice to study loss-of-function effects

  • Conditional knockout models using Cre-loxP systems for tissue-specific deletion

  • Transgenic overexpression models to study gain-of-function effects

• Pharmacological Approaches:

  • Agonistic anti-CD30 antibodies to stimulate signaling

  • Blocking antibodies to inhibit CD30-CD30L interactions

  • Recombinant soluble CD30 as a competitive inhibitor

  • Small molecule inhibitors of downstream signaling components

• Cellular Approaches:

  • Adoptive transfer of CD30-deficient or CD30-overexpressing cells

  • Ex vivo stimulation of CD30 before cell transfer

  • Co-culture systems with CD30L-expressing cells

These approaches can be combined with readouts such as cytokine production, proliferation assays, and in vivo disease models to comprehensively assess CD30's role in immune regulation.

What are the key considerations when designing experiments to study CD30/TNFRSF8 in mouse models of lymphoma and autoimmunity?

When studying CD30/TNFRSF8 in disease models, several factors should be considered:

• Model Selection:

  • For lymphoma studies, choose models that naturally express CD30 (e.g., certain ALK-positive anaplastic large cell lymphoma models)

  • For autoimmunity, consider models where Th2 responses play a significant role

• Timing Considerations:

  • CD30 expression fluctuates during disease progression

  • Temporal analysis at multiple time points is crucial

  • Consider early intervention versus treatment of established disease

• Readouts:

  • Multidimensional assessment including histopathology, flow cytometry, serum biomarkers

  • Functional tests relevant to the specific disease

  • Analysis of both CD30+ cell populations and effects on bystander cells

• Controls:

  • Include CD30-deficient mice on the same background

  • Use isotype controls for antibody treatments

  • Consider sex-specific effects as immune responses may differ

What are common technical challenges when working with mouse CD30/TNFRSF8 in experimental systems and how can they be addressed?

Researchers frequently encounter several challenges when studying mouse CD30/TNFRSF8:

• Low Expression Levels:

  • Solution: Use appropriate stimulation protocols (e.g., ConA, anti-CD3/CD28) to upregulate CD30 expression

  • Employ signal amplification techniques such as tyramine signal amplification for immunohistochemistry

  • Consider enrichment of CD30+ cells prior to analysis

• Antibody Cross-Reactivity:

  • Solution: Validate antibody specificity using CD30 knockout tissues

  • Perform blocking experiments with recombinant CD30 protein

  • Use multiple antibody clones targeting different epitopes

• Protein Degradation:

  • Solution: Add protease inhibitors to all buffers

  • Process samples quickly and maintain cold temperatures

  • For soluble CD30, stabilize with carrier proteins when appropriate

• Glycosylation Variability:

  • Solution: Consider deglycosylation treatments for more consistent molecular weight analysis

  • Use multiple detection methods that are less affected by glycosylation

  • Include controls with known glycosylation patterns

Addressing these challenges requires careful optimization and validation of each experimental protocol for specific applications.

How can researchers optimize detection sensitivity for low-abundance mouse CD30/TNFRSF8 in tissue samples?

Detecting low-abundance CD30/TNFRSF8 requires specialized approaches:

• Sample Preparation Optimization:

  • Fresh tissue preparation whenever possible

  • Optimal fixation protocols to preserve epitopes

  • Antigen retrieval optimization for fixed tissues

• Signal Amplification Techniques:

  • Employ biotin-streptavidin amplification systems

  • Use tyramide signal amplification for immunohistochemistry

  • Consider rolling circle amplification for extremely low abundance targets

• Detection Technology Selection:

  • Choose high-sensitivity ELISA kits with detection limits in the pg/mL range

  • Consider digital ELISA platforms for sub-pg/mL detection

  • Use spectral flow cytometry with bright fluorophores

  • Implement image cytometry for spatial context with high sensitivity

• Enrichment Strategies:

  • Magnetic bead enrichment of CD30+ cells before analysis

  • Laser capture microdissection of regions with expected expression

  • Cell sorting of relevant populations followed by molecular analysis

Combinations of these approaches can significantly improve detection of low-abundance CD30 in complex tissue samples.

How should researchers interpret discrepancies between different detection methods when measuring mouse CD30/TNFRSF8?

When encountering discrepancies between detection methods, consider these analytical approaches:

• Method-Specific Biases:

  • Flow cytometry may detect only cell surface CD30, missing internalized protein

  • ELISA might detect soluble CD30 fragments not recognized by certain antibodies

  • Western blotting can be affected by protein denaturation altering epitope recognition

• Reconciliation Strategies:

  • Use orthogonal methods targeting different epitopes or properties

  • Employ knockout controls to establish true background levels

  • Consider spike-in experiments with recombinant standards

  • Analyze correlation patterns rather than absolute values

• Biological Interpretation:

  • Different forms of CD30 (membrane-bound, soluble, differentially glycosylated) may predominate in different contexts

  • Activation state may affect epitope accessibility

  • Technical variability must be distinguished from biological heterogeneity

What statistical approaches are most appropriate for analyzing mouse CD30/TNFRSF8 expression data across different experimental conditions?

Analyzing CD30/TNFRSF8 expression data requires appropriate statistical methods:

• For Continuous Expression Data (e.g., ELISA, qPCR):

  • Parametric tests (t-test, ANOVA) if normality assumptions are met

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) for non-normal distributions

  • Consider log transformation for skewed distribution common in expression data

  • Use paired tests when comparing the same subjects under different conditions

• For Categorical Data (e.g., percent positive cells):

  • Chi-square or Fisher's exact test for comparing proportions

  • McNemar's test for paired categorical data

• For Complex Experimental Designs:

  • Mixed-effects models for repeated measures with missing data

  • ANCOVA when controlling for covariates

  • Multiple regression to assess relationships between CD30 and multiple factors

• Multiple Testing Correction:

  • Bonferroni correction for strong control of family-wise error rate

  • Benjamini-Hochberg procedure for controlling false discovery rate

  • Consider the biological context when interpreting adjusted p-values

Importantly, power analysis should be conducted during experimental design to ensure sufficient sample sizes for detecting biologically meaningful differences in CD30 expression.