High-throughput measurement of gap junctional intercellular communication

Abstract

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-␣-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 Ͼ90% 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.

Improved multiparametric scrape loading-dye transfer assay for a simultaneous high-throughput analysis of gap junctional intercellular communication, cell density and viability

“…As summarized in the Supplementary Table S3, some of these methods for GJIC assessment have been recently adapted for HTS and/or HCA/HCS. These assays are mostly based on commonly used dye-transfer techniques such as electroporation 21 , laser perforation 22 , microinjection 23 , microfluidic loading 24,25 , parachute/preloading assay [26][27][28] , or on a luminometric or fluorimetric sensing of specific molecules exchanged via gap junctions between donor and recipient cells 29,30 . However, these methods can require a special equipment (electroporating 21 , microscopic 22,27,28 , microinjecting 23 , microfludic 24,25 ), that is not commonly available in a laboratory, and proprietary, closed source software for high-throughput image analysis 21,22,26,28 , or rely on specialized transduced cell models 29,30 .…”

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

“…Individual mNCM in a monolayer of plate cells were injected with pyranine with 8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt dye (Life Technologies) using an in-house developed Robotic Adherent Cell Injection System 68 . Dye transfer measurements were observed 5 min after initial injection 69 .…”

CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures

“…HEK-293 cells were chosen to generate sensor cells since they express moderate levels of gap junctions [35,42]. We tested if GS-293 or EPAC-293 cells could sense the cAMP generated by FSHR-293 cells stimulated by follicle-stimulating hormone (FSH).…”