Real-time electrical measurement of L929 cellular spontaneous and synchronous oscillation

Abstract

Nonexcitable cell types, fibroblasts of heart muscle or astrocytes, are well known for their spontaneous Ca²⁺ oscillations. On the other hand, murine fibroblast (L929) cells are known to be deficient in cell–cell adhesive proteins and therefore lack gap junctions for cellular communication. However, these cells exhibit a unique property of collectively synchronized and spontaneous oscillation, as revealed by real-time monitoring of cells cultured on a 250-μm diameter microelectrode for more than 3 days using an electrical cell-substrate impedance-sensing system (ECIS). Live-cell imaging is a widely used technique for oscillation detection, but it has limitations relating to cellular physiological environment maintenance for microscopic analysis and for prolonged periods of study. The present research emphasizes an electrical-sensing technique (ECIS) capable of overcoming the most important issues inherent in live-cell imaging systems for the detection of L929 cellular spontaneous and synchronized oscillation in real-time for longer periods. Possible mechanisms involved in L929 oscillation were elucidated to be periodic extension/contraction of lamellipodia continued as blebbing, which is produced by signals from the actomyosin complex initiated by connexin hemichannel opening and adenosine triphosphate (ATP) release. By applying the connexin hemichannel inhibitor, flufenamic acid, the hindrance of ATP release and calcium transients were analyzed to elucidate this hypothesis.

Keywords:

• murine fibroblast
• synchronization
• ECIS
• connexin 43
• blebbing
• lamellipodia

Acknowledgements

This work was supported by the WCU Program (R33-2010-000-10067-0) of the Korean Ministry of Education and Science Technology and by the Industrial Strategic Technology Development Program(10035197) funded by the Ministry of Knowledge Economy (MKE, Korea).

Disclosure

The authors report no conflicts of interest in this work.

Supplementary figures

Figure S1 Photograph of eight-well one-electrode (8W1E) ECIS chip.

Note: Right image is the magnification of single well showing the circular detection electrode at the center and common counter electrode.

Abbreviation: ECIS, electric cell-substrate impedance-sensing system.

Figure S2 The whole experiment was repeated thrice and here are the results obtained for two experiments with L929, vero cell, and cell-free medium on a ECIS system.

Note: These results show the consistency of L929 cellular oscillation.

Abbreviation: ECIS, electric cell-substrate impedance-sensing system.

Figure S3 FFT analysis with log frequency of L929 cellular oscillation obtained from ECIS system between 30 to 40 hours.

Abbreviations: ECIS, electric cell-substrate impedance-sensing system; FFT, fast Fourier transform.

Figure S4 FFT analysis with log frequency of L929 cellular oscillation obtained from ECIS system between 55 to 70 hours.

Abbreviations: ECIS, electric cell-substrate impedance-sensing system; FFT, fast Fourier transform.