Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons
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.
Key wordsFura-2 AM Ratiometric calcium indicators Thapsigargin SOCE Progenitors Calcium mapping Calcium indicators Stem cells Neural differentiation
We are grateful to Central Imaging and Flow Cytometry Facility, National Centre for Biological Sciences (NCBS), TIFR for the use of microscopes and Stem Cell Facility for the infrastructure for hESC and NPC work. This work was funded by a grant from the Department of Biotechnology, Ministry of Science and Technology, Govt. of India (BT/PR6371/COE/34/19/2013) and core funds from NCBS, TIFR, to GH. Financial support from the DBT-RA program in Biotechnology and Life Sciences, Govt. of India to RG and INSPIRE fellowship under DST, Govt. of India to PC are gratefully acknowledged.
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