Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin

Area of Science:

• Biophysics
• Cell Biology
• Biotechnology

Background:

• Mechanosensitive proteins are vital for physiological processes like hearing and blood flow regulation.
• Accurately applying mechanical forces to cells for studying mechanosensitivity is challenging.
• Existing methods lack the precision and reproducibility needed for quantitative assessment.

Purpose of the Study:

• To introduce a novel device for controlled, flow-based mechanical stimulation of cells in a 96-well plate format.
• To enable quantitative assessment of mechanosensitive protein function under defined mechanical forces.
• To investigate the role of the mechanosensitive protein GPR68 in cellular responses to mechanical stimuli.

Main Methods:

• Development of a 96-well plate-compatible device for generating controlled shear-force stimulation.
• Mechanical stimulation of HEK 293T cells expressing the G protein-coupled receptor GPR68.
• Assaying intracellular calcium levels using a fluorescence plate reader to measure GPR68 signaling.

Main Results:

• The novel device allows for highly-controlled, reproducible mechanical stimulation of cells.
• GPR68 signaling in HEK 293T cells was found to be dependent on the applied shear force.
• Intracellular calcium levels directly correlated with the mechanical forces applied.

Conclusions:

• The developed device is a valuable tool for quantitative assessment of mechanotransduction.
• This method provides a new way to study the function of mechanosensitive proteins like GPR68.
• The findings highlight the direct relationship between mechanical force and GPR68-mediated signaling pathways.