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Methods Mol Biol. 2019;2029:257-271. doi: 10.1007/978-1-4939-9631-5_20.

Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons.

Author information

1
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.
2
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India. gaiti@ncbs.res.in.

Abstract

Calcium imaging in an ex-vivo setup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380 nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the endoplasmic reticulum (ER)-driven store-operated calcium entry (SOCE) in human neural precursors (NPCs) and spontaneously differentiated neurons derived from a pluripotent human embryonic stem cell (hESC) line. The derivation of neural precursors from stem cells and their subsequent spontaneous neural differentiation is also explained. The method can be used for various non-excitable and excitable cell types including neurons, be it freshly isolated, from frozen vials, or derived from different stem cell lines.

KEYWORDS:

Calcium indicators; Calcium mapping; Fura-2 AM; Neural differentiation; Progenitors; Ratiometric calcium indicators; SOCE; Stem cells; Thapsigargin

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