A versatile, bar-coded nuclear marker/reporter for live cell fluorescent and multiplexed high content imaging

Area of Science:

• Cell Biology
• Biotechnology
• High-Throughput Screening

Background:

• Current high-throughput screening methods often rely on single cellular or biochemical outputs, limiting biological relevance.
• High-throughput, multi-channel fluorescence microscopy allows quantification of multiple outputs but is constrained by the limited number of distinct fluorescent reporters.

Purpose of the Study:

• To develop and validate a novel cellular barcoding technology for combining and distinguishing multiple cell-based assays within a single well.
• To enable simultaneous analysis of multiple assay outputs for more biologically relevant screening and complex cellular system modeling.

Main Methods:

• Developed assay-specific fluorescent markers using combinations of red fluorescent proteins and a nuclear localization signal.
• These markers are excited by a common wavelength but distinguished ratiometrically by their fluorescence in two emission channels.
• Validated the technology by co-plating three distinct cell-based assays and confirming retention of drug responses using standard image analysis.

Main Results:

• Successfully demonstrated the ability to distinguish multiple assays within the same well using the cellular barcoding system.
• Validated that drug responses for individual assays are preserved when performed concurrently using the barcoding method.
• Showcased the potential for separating individual cells expressing distinguishable bar-code markers via image analysis.

Conclusions:

• The cellular barcoding technology enables simultaneous analysis of multiple assays, enhancing the biological relevance of screening.
• This approach facilitates the identification and characterization of hits based on multiple criteria, improving drug discovery.
• The technology supports the recreation of complex cell mixtures, a key area for advancing various biological research fields.