High-throughput measurement of gap junctional intercellular communication

Area of Science:

• Cell Biology
• Biophysics
• Biotechnology

Background:

• Gap junctional intercellular communication (GJIC) is vital for cell-to-cell electrical propagation.
• Disruptions in GJIC are implicated in cardiac arrhythmias.
• Conventional microinjection techniques for measuring GJIC are labor-intensive and low-throughput.

Purpose of the Study:

• To develop and validate a robotic microinjection system for high-throughput GJIC assessment.
• To evaluate the system's performance in terms of speed, success rate, and cell viability.
• To quantify GJIC in different cell lines and assess the effects of an inhibitor.

Main Methods:

• Development of a highly automated robotic microinjection system for cell culture.
• Evaluation of GJIC using dye transfer assays in HeLa, HEK293, and HL-1 cell lines.
• Quantification of GJIC inhibition using 18-α-glycyrrhetinic acid.
• Real-time monitoring of dye transfer for molecular diffusion coefficient calculation.

Main Results:

• The robotic system achieved an injection speed of 22.7 cells/min with a 95% success rate and >90% cell viability.
• Dye transfer correlated with cell type-specific connexin expression.
• GJIC inhibition was dose-dependent on the applied inhibitor.
• Molecular diffusion coefficients were calculated via real-time dye transfer monitoring.

Conclusions:

• The robotic microinjection system significantly enhances the throughput and automation of GJIC measurements.
• This technique allows for rapid and reliable assessment of gap junction function in various cell types.
• The system provides a valuable tool for studying GJIC in physiological and pathological conditions, including cardiac arrhythmias.