3 January 2014 Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching
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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.
© 2014 Society of Photo-Optical Instrumentation Engineers (SPIE)
Maria Kuzma-Kuzniarska, Maria Kuzma-Kuzniarska, Clarence Yapp, Clarence Yapp, Thomas W. Pearson-Jones, Thomas W. Pearson-Jones, Andrew K. Jones, Andrew K. Jones, Philippa A. Hulley, 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 . Submission:
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