NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

Overview

This protocol allows for the rapid assessment of NF-魏B activation in cell lines using an NF-魏B::luciferase reporter. It also includes gene expression analysis via RT-qPCR in cells infected with Salmonella Typhimurium.

Key Study Components

Area of Science

• Cell Biology
• Immunology
• Microbiology

Background

• NF-魏B is a crucial transcription factor involved in various cellular processes.
• It plays a significant role in inflammatory responses by regulating cytokine and chemokine expression.
• This study focuses on NF-魏B activation induced by Salmonella Typhimurium.
• The HeLa cell line is utilized for this research due to its stable transfection with an NF-魏B luciferase reporter.

Purpose of Study

• To measure NF-魏B activation in response to different stimuli.
• To explore the effects of Salmonella Typhimurium on NF-魏B signaling.
• To facilitate high-throughput screening of factors influencing NF-魏B activation.

Methods Used

• Measurement of luminescence in cell lysates to assess NF-魏B activation.
• RT-qPCR for gene expression analysis in infected cells.
• Use of HeLa cells stably expressing NF-魏B::luciferase reporter constructs.
• High-throughput screening techniques to evaluate various conditions.

Main Results

• Successful measurement of NF-魏B activation levels in response to Salmonella infection.
• Identification of key factors that modulate NF-魏B signaling pathways.
• Demonstrated the effectiveness of the luciferase reporter system for high-throughput assays.
• Provided insights into the role of NF-魏B in inflammatory responses.

Conclusions

• The protocol offers a reliable method for studying NF-魏B activation.
• It can be applied to various research contexts involving inflammation and infection.
• This approach enhances our understanding of NF-魏B's role in cellular responses to pathogens.

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