Intracellular calcium recording and immunostaining of single neural populations in vitro

Abstract

Calcium plays a pivotal role as an intracellular messenger, eliciting a diverse array of cellular responses. One of the most common calcium-sensitive fluorescent indicators is the ratiometric dye Fura-2. The primary advantage of using ratiometric dyes lies in their independence from factors, such as illumination intensity, photobleaching, dye concentration, and focal alterations, among others. This independence allows for the determination of intracellular calcium concentration free from these interfering variables. In this protocol, we describe the utilization of Fura-2 to assess intracellular calcium elevations in cultures of neural stem cells using video-microscopy equipment. We have previously applied this methodology, commonly referred to as calcium imaging, to investigate intracellular calcium dynamics triggered by the activation of purinergic receptors in a variety of neural populations (Gomez-Villafuertes et al., Cell Transplant 24(8):1493–1509, 2015; Hervas et al., J Neurosci Res 73(3):384–399, 2003; Diaz-Hernandez et al., J Cell Sci 121(Pt 22): 3717–3728, 2008; Carrasquero et al., J Neurochem 110(3):879–889, 2009; Gomez Villafuertes et al., FEBS J 276(18):5307–5325, 2009).