All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

作者： Qin Luo*1, Miao Huang*2, Chenyu Liang2, Justin Zhang3, Gaoming Lin1, Sydney Yu1, Mai Tanaka4,5, Sharon Lepler4,5, Juan Guan5,6,7, Dietmar Siemann4,5, Xin Tang2,5 1Department of Electrical and Computer Engineering, Herbert Wertheim College of Engineering,University of Florida, 2Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering,University of Florida, 3Department of Electrical and Computer Engineering,University of California, 4Department of Radiation Oncology, College of Medicine,University of Florida, 5UF Health Cancer Center,University of Florida, 6Department of Physics, College of Liberal Arts and Sciences,University of Florida, 7Department of Anatomy and Cell Biology, College of Medicine,University of Florida

简介：

Overview

This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP). This method illustrates molecular mechanisms of mechanobiology and optical electrophysiology using automatic live-cell imaging techniques.

Key Study Components

Area of Science

• Mechanobiology
• Optical Electrophysiology
• Live-Cell Imaging

Background

• This protocol enables automatic, multifunctional, high-throughput, self-drift correcting, and long-term image acquisition.
• It is compatible with most fluorescent microscopic platforms, such as NYCOM.
• Understanding the function of each step is crucial for successful experimentation.
• The protocol involves precise environmental control and imaging techniques.

Purpose of Study

• To elucidate the mechano-sensitivity of Yes-associated protein (YAP).
• To provide a comprehensive guide for researchers to implement advanced imaging techniques.
• To enhance understanding of molecular mechanisms in mechanobiology.

Methods Used

• Setting up an environment chamber on a motorized stage of an inverted microscope.
• Adjusting CO2 flow rate and temperature for optimal cell conditions.
• Utilizing Micro Manager for cell observation and imaging.
• Implementing a macro for automated imaging and data acquisition.

Main Results

• Successful attachment and imaging of cells under controlled conditions.
• Accurate acquisition of multi-channel images for mechanobiological analysis.
• Demonstration of the protocol's effectiveness in elucidating YAP mechano-sensitivity.
• Validation of the method across various cell types.

Conclusions

• The protocol provides a robust framework for mechanobiological studies.
• It facilitates high-throughput imaging and analysis of cellular responses.
• Future applications may extend to various biological phenomena involving mechanobiology.

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