Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

Maria Kuzma-Kuzniarska; Clarence Yapp; Thomas W. Pearson-Jones; Andrew K. Jones; Philippa A. Hulley

doi:10.1117/1.JBO.19.1.015001

3 January 2014 Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

Maria Kuzma-Kuzniarska, Clarence Yapp, Thomas W. Pearson-Jones, Andrew K. Jones, Philippa A. Hulley

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Journal of Biomedical Optics, Vol. 19, Issue 1, 015001 (January 2014). https://doi.org/10.1117/1.JBO.19.1.015001

Abstract

Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β -glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.

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Maria Kuzma-Kuzniarska, Clarence Yapp, Thomas W. Pearson-Jones, Andrew K. Jones, and Philippa A. Hulley "Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching," Journal of Biomedical Optics 19(1), 015001 (3 January 2014). https://doi.org/10.1117/1.JBO.19.1.015001

Published: 3 January 2014

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