TNFA Mouse, Sf9

What is TNFA Mouse, Sf9 and how is it structurally characterized?

TNFA Mouse, Sf9 is a recombinant mouse TNF-α protein produced in Sf9 Baculovirus cells. It is a single, glycosylated polypeptide chain containing 162 amino acids (80-235 a.a.) with a molecular mass of 18kDa, though it typically appears at approximately 18-28kDa on SDS-PAGE due to glycosylation patterns. The protein is expressed with a 6 amino acid His tag at the C-Terminus and is purified through proprietary chromatographic techniques .

What are the known biological functions of TNF-α in mouse models?

TNF-α serves multiple crucial functions in mouse models:

• Regulation of immune cells with primary involvement in inflammatory responses

• Induction of apoptotic cell death

• Promotion of cellular proliferation and differentiation

• Involvement in tumorigenesis and viral replication

• Participation in lipid metabolism and coagulation processes

Research with TNF-deficient mice has demonstrated that TNF-α is essential for normal responses to infection, as these mice are highly susceptible to challenges with infectious agents like Candida albicans and show deficiencies in granuloma development. Additionally, they fail to form germinal centers after immunization, indicating TNF-α's important role in adaptive immunity .

How should TNFA Mouse, Sf9 be stored for optimal stability?

For optimal stability, TNFA Mouse, Sf9 should be stored at 4°C if the entire vial will be used within 2-4 weeks. For longer periods, it should be stored frozen at -20°C or preferably at -80°C. When aliquoting the protein, storage at -80°C is recommended for extended shelf life (up to 1 year from the date of receipt). The protein remains stable at -20°C for approximately 3 months after opening. It's critical to avoid repeated freeze-thaw cycles as these can significantly reduce protein activity .

How can researchers effectively measure mouse TNF-α levels in experimental samples?

Several validated methods can be employed to measure mouse TNF-α levels:

ELISA (Enzyme-Linked Immunosorbent Assay):

• The Quantikine Mouse TNF-alpha Immunoassay offers a 4.5-hour solid-phase ELISA designed for mouse TNF-α detection in cell culture supernatants, serum, and plasma

• Sensitivity levels of approximately 9.1 pg/ml can be achieved with high-quality ELISA kits

• The detection range typically spans from approximately 46.88 pg/ml to 3000 pg/ml

HTRF (Homogeneous Time-Resolved Fluorescence):

• Requires smaller sample volumes (≈16 μL)

• Provides results in approximately 1 hour at room temperature

• Offers a detection range of 20-6,000 pg/ml with LOD (Limit of Detection) at 3 pg/ml

These methods show good precision with intra-assay CV% as low as 2-4% and inter-assay CV% of 6-9%, depending on the specific kit used .

What experimental controls should be included when working with TNFA Mouse, Sf9 in cell culture studies?

For rigorous cell culture experiments with TNFA Mouse, Sf9, researchers should implement the following controls:

• Vehicle control: Cells treated with the same buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 10% glycerol) without the protein

• Dose-response controls: Multiple concentrations of TNFA Mouse, Sf9 to establish dose-dependent effects

• Time-course controls: Measurements at different time points to determine optimal response timing

• TNF-α neutralizing antibody controls: To confirm specificity of observed effects

• Positive controls: Use of known TNF-α inducers like LPS in parallel experiments

• Unstimulated cell controls: Baseline measurements for comparison

• Cross-reactivity controls: Testing with recombinant human TNF-α to check species specificity

• Cell viability controls: To distinguish between cytotoxic effects and specific TNF-α signaling

For studies involving microglial activation or inflammation models, RAW 264.7 cells have been successfully used as model systems with LPS stimulation serving as a positive control .

How does TNFA Mouse, Sf9 compare with other TNF-α expression systems in functional studies?

TNFA Mouse, Sf9 (baculovirus-expressed) offers distinct advantages and considerations compared to other expression systems:

For cytotoxicity assays using HT-29 human colon adenocarcinoma cells, the effective dose (ED50) of similar TNF superfamily proteins from Sf9 has been established at around 2.866 µg/mL when used with a cross-linking antibody (anti-polyHistidine) and recombinant human IFN-gamma .

What are the key considerations when using TNFA Mouse, Sf9 in neuroinflammation models?

When employing TNFA Mouse, Sf9 in neuroinflammation studies, researchers should consider:

• Dosage determination: Peripheral administration of TNF-α (intraperitoneal injection) in mice can increase serum and brain levels of proinflammatory mediators including TNF-α, IL-6, and MCP-1 in a dose- and time-dependent manner. Studies have demonstrated that a single injection can induce neuroinflammation .

• Blood-brain barrier (BBB) considerations: While peripheral TNF-α does not readily cross the intact BBB, it can affect central nervous system function through:

  • Activation of BBB endothelial cells

  • Induction of cytokine release at the BBB interface

  • Signal transduction via vagal afferents

• Behavioral assessments: TNF-α administration induces robust sickness behavior characterized by:

  • Reduced locomotor activity

  • Decreased fluid intake

  • Body weight loss

  These effects must be distinguished from true depressive-like behaviors in experimental designs .

• Glial cell activation: TNF-α challenge leads to increased astrocyte activation (measurable in Gfap-luc mice) and elevated immunoreactivity against microglial markers like Iba1, particularly in brain regions such as the dentate gyrus .

• Transgenic model integration: Studies involving 3xTg-AD mice (Alzheimer's model) have revealed that neuronal TNF-α expression promotes inflammation and neuronal cell death. Consider using appropriate transgenic models to study specific disease contexts .

How can researchers differentiate between the roles of TNF-α receptors (TNFR1 vs. TNFR2) when using TNFA Mouse, Sf9?

To differentiate between TNFR1 and TNFR2 signaling pathways using TNFA Mouse, Sf9:

• Utilize receptor-specific knockout models:

  • TNF-R1(-/-) mice show impairments in learning and memory in the Barnes maze and Y-maze

  • TNF-R2(-/-) mice display good memory but slow learning in the same tests

  • TNF-R2(-/-) mice exhibit decreased anxiety-like behavior compared to wild-type mice

• Employ selective receptor agonists alongside TNFA Mouse, Sf9:

  • Use receptor-specific antibodies or engineered variants that preferentially activate either TNFR1 or TNFR2

  • Compare responses to broad TNF-α stimulation versus receptor-specific activation

• Implement receptor neutralization strategies:

  • Apply receptor-specific neutralizing antibodies prior to TNFA Mouse, Sf9 treatment

  • Use soluble receptor fragments (like etanercept for TNFR2) to differentially block receptor activation

• Analyze downstream signaling pathways:

  • TNFR1 primarily activates proinflammatory and apoptotic pathways through TRADD and FADD

  • TNFR2 predominantly mediates cell survival, proliferation, and tissue regeneration through TRAF2

These approaches have demonstrated that both receptors play roles in normal CNS function, with knockout of either receptor impairing cognitive functions, though through potentially different mechanisms .

What are the optimal cell culture conditions for measuring TNFA Mouse, Sf9 biological activity?

For robust measurement of TNFA Mouse, Sf9 biological activity:

• Cell line selection:

  • L929 cells are commonly used for TNF-α cytotoxicity assays

  • RAW 264.7 macrophages serve as excellent models for TNF-α production and response studies

  • HT-29 human colon adenocarcinoma cells in combination with cross-linking antibodies and IFN-gamma

• Culture conditions:

  • For RAW 264.7 cells: Culture in HGDMEM with 100 μg/mL Kanamycin and 2 mM L-glutamine

  • Starvation period: 24 hours before treatment to synchronize cells

  • Treatment parameters: When using LPS as positive control, 5 μg/mL concentration is effective

• Activity detection methods:

  • MTT assay for cell viability assessment following TNF-α treatment

  • Flow cytometry using fluorescent-conjugated antibodies (Alexa Fluor 488 goat anti-mouse IgG for mouse TNF-α detection)

• Response quantification:

  • In LPS-stimulated RAW 264.7 cells, TNF-α levels typically reach approximately 6346.4 pg/mL compared to 83.7 pg/mL in unstimulated cells

  • TNF-α biological activity in cytotoxicity assays typically shows ED50 values in the range of 2-3 μg/mL .

What approaches can address cross-reactivity concerns when working with TNFA Mouse, Sf9 in mixed-species experimental systems?

When addressing cross-reactivity concerns in mixed-species systems:

• Species specificity evaluation:

  • Mouse TNF-α ELISA kits show minimal cross-reactivity with other cytokines like TNF RI, TNF RII, OPG, CD40 receptor, IL-1 beta, IL-16, and IL-5

  • With human TNF-α, approximately 3.0% cross-reactivity has been observed (with a standard deviation of 0.7%)

• Pre-experimental validation:

  • Test the reactivity of TNFA Mouse, Sf9 on both mouse and non-mouse cell lines

  • Compare response patterns using species-specific detection systems

  • Conduct preliminary dose-response studies to identify potential threshold differences

• Neutralization controls:

  • Use species-specific neutralizing antibodies to confirm attribution of observed effects

  • Include parallel experiments with human and mouse TNF-α to establish relative potencies

• Modified experimental design:

  • For humanized antibody development (like anti-TNF-α therapeutics), consider the structural differences between species

  • When humanizing murine antibodies against TNF-α, careful selection of framework residues is critical to preserve antigen binding affinity .

How can researchers troubleshoot inconsistent results when using TNFA Mouse, Sf9 in inflammasome activation studies?

When troubleshooting inconsistent results in inflammasome studies:

• Protein quality assessment:

  • Verify protein activity using a standardized cytotoxicity assay

  • Check for potential degradation via Western blot analysis

  • Assess glycosylation status, as variations can affect activity

• Experimental design optimization:

  • For NLRP3 inflammasome studies, consider that both Aβ and ATP can activate NLRP3 via P2X7R in microglia

  • In 5xFAD mouse models, closely monitor treatment timing and dosing regimens

• Technical considerations:

  • For proteomic analyses of mouse brains treated with immunomodulators, include appropriate controls

  • When analyzing behavioral outcomes, use multiple tests (Morris water maze, Y-maze, novel object recognition) to comprehensively assess cognitive effects

• Cell-specific responses:

  • TNF-α effects on neuroinflammation may vary between microglia, astrocytes, and neurons

  • For studies in Sf9 cells themselves, note that apoptosis-related genes (97 putative genes identified through transcriptome analysis) can be significantly modulated

• Data normalization strategies:

  • When analyzing cytokine production, normalize to total protein content

  • For behavioral studies, account for baseline differences between experimental groups .

How do findings from TNFA Mouse, Sf9 research translate to understanding human inflammatory disorders?

While TNFA Mouse, Sf9 is a research tool for mouse models, its applications provide valuable insights into human inflammatory disorders:

• Therapeutic target validation:

  • TNF-α blockade is an effective treatment for rheumatoid arthritis (RA) and other inflammatory diseases

  • Different TNF-α blockade strategies (etanercept vs. TNF-α kinoid vaccine) show distinct profiles in infection susceptibility, informing human therapeutic approaches

• Neuroinflammatory disease insights:

  • In Alzheimer's disease models (3xTg-AD mice), neuronal TNF-α expression promotes inflammation and neuronal death

  • These findings correlate with observations that TNF-α system activation may contribute to inflammation-associated depression in humans

• Infection resistance considerations:

  • TNF-α blockade in humans is associated with reduced resistance to Mycobacterium tuberculosis and Listeria monocytogenes

  • Mouse models using TNFA Mouse, Sf9 help predict and understand these susceptibilities

• Developmental insights:

  • Studies in TNF-deficient mice show that they develop normally but exhibit specific immunological deficiencies

  • This provides context for understanding the role of TNF-α in human development and immune system maturation .

What are the key methodological differences when comparing TNF-α neutralization strategies in mouse models versus clinical applications?

When comparing TNF-α neutralization approaches:

Research demonstrates that TNF-α kinoid vaccine approaches may allow for better remaining host defense than soluble receptor strategies like etanercept, suggesting potential for improved safety profiles in clinical applications .

How can TNFA Mouse, Sf9 be utilized in developing and testing novel anti-inflammatory therapeutics?

TNFA Mouse, Sf9 serves as a valuable tool in anti-inflammatory therapeutic development:

• Target validation studies:

  • Assess whether novel compounds can effectively neutralize or modulate TNF-α activity

  • Determine target selectivity by comparing effects against other cytokines

• Screening assays:

  • Develop cell-based assays using TNFA Mouse, Sf9 as a stimulant

  • Screen compound libraries for molecules that inhibit TNF-α-induced cellular responses

• Mechanism of action studies:

  • Compare direct TNF-α inhibitors versus modulators of downstream signaling

  • Distinguish between TNFR1 and TNFR2 pathway interventions

  • Evaluate effects on specific inflammatory mechanisms (e.g., NLRP3 inflammasome)

• Pharmacological validation:

  • Test dose-response relationships in vitro before advancing to in vivo studies

  • Assess compound stability and activity in physiologically relevant conditions

• Specialized applications:

  • For Alzheimer's disease research, compounds like TDCA (a GPCR19 agonist) can be evaluated for their ability to modulate TNF-α effects in 5xFAD mouse models

  • In inflammatory bowel disease models, TNF-α blockade strategies can be compared for efficacy and safety

Results from such studies have demonstrated that different anti-TNF strategies (e.g., kinoid vaccines vs. soluble receptors) can yield distinct efficacy and safety profiles, informing translational development paths .