High-throughput measurement of gap junctional intercellular communication.
Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.
Advanced Micro and Nanosystems Laboratory, University of Toronto, Toronto, Ontario, Canada; and.
Jun
J
Liu
Division of Cardiology, the Hospital of Sick Children, Toronto, Ontario, Canada.
Vinayakumar
V
Siragam
Advanced Micro and Nanosystems Laboratory, University of Toronto, Toronto, Ontario, Canada; and.
Jun
J
Chen
Division of Cardiology, the Hospital of Sick Children, Toronto, Ontario, Canada.
Michael D
MD
Fridman
Division of Cardiology, the Hospital of Sick Children, Toronto, Ontario, Canada.
Robert M
RM
Hamilton
Advanced Micro and Nanosystems Laboratory, University of Toronto, Toronto, Ontario, Canada; and sun@mie.utoronto.ca.
Yu
Y
Sun
D000818:Animals; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D005456:Fluorescent Dyes / Q000008:administration & dosage; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology*; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D057809:HEK293 Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006367:HeLa Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D008845:Microinjections; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D012371:Robotics; D013997:Time Factors
D005456:Fluorescent Dyes; D006034:Glycyrrhetinic Acid
automation; dye transfer; gap junctional intercellular communication; robotic microinjection