The Analysis of Gap Junctional Intercellular Communication Among Osteocytes in Chick Calvariae by Fluorescence Recovery
This chapter describes the use of fluorescence recovery after photobleaching (FRAP) for analyzing gap junctional intercellular communication (GJIC) among osteocytes in chick calvariae by confocal laser scanning microscope.
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The Analysis of Gap Junctional Intercellular Communication Among Osteocytes in Chick Calvariae by Fluorescence Recovery After Photobleaching Ziyi Wang, Yoshihito Ishihara, and Hiroshi Kamioka Abstract This chapter describes the use of fluorescence recovery after photobleaching (FRAP) for analyzing gap junctional intercellular communication (GJIC) among osteocytes in chick calvariae by confocal laser scanning microscope. Keywords Osteocytes, Gap-junctional intercellular communication, Fluorescence recovery after photobleaching, One-phase exponential association equation
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Introduction Cell-cell communication is crucial for the cell as a member of a tissue or an organ. Osteocytes are the most abundant cells in bone, forming a three-dimensional (3D) structural network by connecting with each other through long, slender cell processes. They help contribute to synthesize, resorb, and repair skeletal tissue by directing the differentiation and activity of surface osteoblasts and osteoclasts. Blockade of these networks among bone cells diminishes the bone quality and leads to skeletal fragility [1].
1.1 Osteocyte Network
Gap junctional intercellular communications (GJICs) play an important role in which bone cells coordinate their actions. Small ions, molecules, and second messengers can be directly exchanged between bone cells via the aqueous channels formed by gap junctions. Therefore, GJICs form an efficient interconnected cellular network that results in the functional and efficient coordination among bone cells. The gap junctions presented in bone cells are able to sense forces, since the mechanical simulation of osteocytes increases the expression of connexins in vitro and in vivo, presumably creating enhanced connections with neighboring cells to strengthen the transmission of mechanical information within the osteocyte network [2]. Our previous study showed that extracellular pH, extracellular calcium, and parathyroid hormone
Ziyi Wang et al.
significantly altered the rate of fluorescence recovery after photobleaching [3]. Indeed, the maturation of osteocytes affects the rate of recovery of fluorescence, and the speed of dye diffusion may differ as well [4]. 1.2 Fluorescence Recovery After Photobleaching
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Compared with the use of fluorescence dye via microinjection or scrape loading, fluorescence recovery after photobleaching (FRAP) is noninvasive and easier and faster to perform. In the FRAP technique, we use the chemical and biophysical properties (such as the size) of tracers without a metabolic role to define and quantify the communication capacity. In general, it was assumed that the choice of dye would not significantly influence the fluorescence redistribution. A previous study showed that the rate of fluorescence recovery did not differ significantly among different dyes, even when their fluorescence kinetic profiles were obviously different [5].
Materials Animals
Lohmann LSL-classic embryonic chicks were obtained from Lohmann Tierzucht (Cuxhaven, Germany).
2.2 General Reagents
1. Culture medi
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