High-throughput measurement of gap junctional intercellular communication

• Published in: American journal of physiology. Heart and circulatory physiology 2014-06, Vol.306 (12), p.H1708-H1713
• Main Authors: Liu, Jun, Siragam, Vinayakumar, Chen, Jun, Fridman, Michael D, Hamilton, Robert M, Sun, Yu
• Format: Article
• Language: English
• Subjects: Animals; Cardiac arrhythmia; Cell Communication - drug effects; Cell Communication - physiology; Cell Survival - physiology; Cells; Density; Fluorescent Dyes - administration & dosage; Gap Junctions - drug effects; Gap Junctions - physiology; Glycyrrhetinic Acid - pharmacology; HEK293 Cells - cytology; HEK293 Cells - drug effects; HEK293 Cells - physiology; HeLa Cells - cytology; HeLa Cells - drug effects; HeLa Cells - physiology; High-Throughput Screening Assays - methods; Humans; Mice; Microinjections; Myocytes, Cardiac - cytology; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - physiology; Propagation; Robotics; Signal transduction; Time Factors
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00110.2014

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.