Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain

Area of Science:

• Biochemistry
• Cell Biology
• Immunology

Background:

• Tumor necrosis factor-alpha (TNF-alpha) is a key cytokine in inflammation and immunity.
• Accurate measurement of TNF-alpha biological activity is crucial for research and drug development.
• Existing assays may lack sensitivity or require complex procedures.

Purpose of the Study:

• To develop a simple, sensitive, and robust fluorescence-based assay for quantifying TNF-alpha biological activity.
• To utilize SYTOX Green nucleic acid stain for detecting TNF-induced cell necrosis.
• To establish a high-throughput screening method for TNF-alpha and its modulators.

Main Methods:

• A microplate-based assay using SYTOX Green stain to detect cell necrosis induced by TNF-alpha in actinomycin D-sensitized cell lines (WEHI 164, WEHI 13var, L929).
• Monitoring fluorescence enhancement upon SYTOX Green binding to nucleic acids in cells with compromised membranes.
• Single-step reagent addition followed by a 6-8 hour incubation without wash steps.

Main Results:

• The assay detected as little as 2.5 pg/mL and quantified 25-500 pg/mL of recombinant murine and human TNF-alpha.
• Demonstrated complete inhibition of TNF-alpha activity using a monoclonal anti-TNF-alpha antibody.
• Determined that approximately 20 ng/mL of antibody neutralized 50% of TNF-alpha activity.

Conclusions:

• The developed assay is simple, sensitive, and suitable for measuring TNF-alpha biological activity.
• The assay is readily adaptable for automation and high-throughput screening of TNF-alpha and its inhibitors.
• This method provides a valuable tool for immunological and cell-based research involving TNF-alpha.