Screening protocol for the identification of modulators by immunofluorescent cell-based assay

Area of Science:

• Biochemistry
• Cell Biology
• Drug Discovery

Background:

• High-throughput assays are crucial for identifying compounds that modulate cellular activity.
• Quantifying target protein modulation often involves complex imaging and analysis.

Purpose of the Study:

• To present an automated analysis platform for quantifying nuclear foci to assess compound inhibition.
• To integrate cell-based assays, high-content screening, and confocal microscopy with automated statistical analysis.

Main Methods:

• Developed an automated platform combining cell-based assays, high-content screening, and confocal microscopy.
• Implemented a web-based tool using Pipeline Pilot and R for statistical analysis.
• Included positive and negative controls and calculated Z-prime and Window values for assay reliability.

Main Results:

• The platform quantifies nuclear foci, indicating compound inhibitory effects on target proteins.
• It computes inhibition values based on various parameters (e.g., foci number, intensity, area).
• Provides outlier detection, filtering, and comprehensive statistical plots for result interpretation.

Conclusions:

• The automated platform offers a fast, user-friendly, and reliable method for analyzing compound inhibition via nuclear foci.
• It facilitates efficient exploration of screening data, aiding in the interpretation of assay results.
• This system enhances the process of identifying potential therapeutic compounds in drug discovery pipelines.