High Throughput Assay of Diffusion through Cx43 Gap Junction Channels with a Microfluidic Chip

Abstract

This paper describes a microfluidic-based assay capable of measuring gap-junction mediated dye diffusion in cultured cells. The technique exploits multi-stream laminar flow to selectively expose cells to different environments, enabling continuous loading of cells in one compartment while monitoring, in real time, dye diffusion into cells of a neighboring compartment. A simple one dimensional diffusion model fit to the data extracted the diffusion coefficient of four different dyes, 5-(6)-carboxyfluorescein (CFDA), 5-chloromethylfluorescein (CMFDA), Oregon green 488 carboxylic acid and calcein. Different inhibitors were assayed for their ability to reduce dye coupling. The chip can screen multiple inhibitors in parallel in the same cell preparation, demonstrating its potential for high throughput. The technique provides a convenient method to measure gap junction mediated diffusion and a screen for drugs that affect gap junction communication.

High-throughput measurement of gap junctional intercellular communication

“…The diffusion coefficients of control group and 50-M treatment group were 3.62ϫ10 Ϫ7 and 1.28ϫ10 Ϫ7 cm 2 /s, respectively. These diffusion coefficients are within the same order of magnitude as previously reported values for fluorescent dyes with similar molecular mass (3,31,39). The diffusion coefficient differences also indicate that molecule diffusion through gap junctions was suppressed by 18-␣GA treatment.…”

A microfluidic platform for measuring electrical activity across cells

“…This approach allows observation of dye loading and diffusion between different regions. 25,26 Here, we show that the tri-stream chip can be used to measure electrical coupling across gap junctions. The chip is designed in a generic form so that it can be used to measure both electrical conductance and molecular diffusion in real time.…”

“…These optimal chamber and flow parameters were explored using computational fluid dynamics method as described previously. 25 …”

“…We have previously characterized the flow stability and the boundary width of the central stream using fluorescence tracers and finite element simulations in a cell-free chamber. 25 Figure 2(a) shows a control trace of the electrical resistance across the sensing chamber (without cells) when the sucrose solution, with and without the gap junction blocker (2-APB), was perfused intermittently in the middle stream of the chip. It shows that a stable sucrose gap can be created repeatedly.…”

“…5(b)). 25 2-APB (400 lM) was applied for the durations indicated by double headed arrows in Figs. 5(a) and 5(c); the change in resistance was measured simultaneously and is plotted in Fig.…”