Case study on live cell apoptosis-assay using lamin-chromobody cell-lines for high-content analysis

Area of Science:

• Cell Biology
• Biotechnology
• Immunology

Background:

• Understanding cellular processes requires detailed data on endogenous components.
• Current methods like fluorescent fusion proteins lack endogenous context, while antibodies typically require fixed cells.
• A need exists for technologies that visualize endogenous molecules in living cells.

Purpose of the Study:

• To introduce and validate chromobodies as a novel tool for detecting and tracing endogenous cellular components in vivo.
• To demonstrate the application of chromobodies for real-time monitoring of cellular events, such as apoptosis.

Main Methods:

• Utilizing the antigen-binding domain of heavy-chain antibodies (VHH) from Camelidae.
• Fusing VHH domains with fluorescent proteins to create functional chromobodies.
• Expressing chromobodies in living cells for in vivo detection and tracking.

Main Results:

• Demonstrated successful expression of VHH-based fluorescent chromobodies in living cells.
• Showcased chromobodies' ability to detect and trace endogenous proteins and other cellular components in vivo.
• Validated chromobody utility for real-time monitoring of apoptosis in living cells.

Conclusions:

• Chromobodies represent a significant advancement for high-content cellular analysis.
• This technology offers expanded capabilities for studying cellular dynamics and physiopathology in real time.
• Chromobodies can detect diverse antigenic structures, including posttranslational modifications and nonprotein components.