ABSTRACT
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As discussed throughout this book, the development of calcium-sensitive indicators
based on the calcium chelator BAPTA revolutionized the study of cellular calcium
homeostasis (Chapter 1). The first two decades of the assessment of cytoplasmic
calcium ([Ca2þ]CYT) using calcium-sensitive fluorescence indicators (such as fluo-3 and fura-2) were dominated by studies involving cuvette-and microscope-based
equipment. Critically, the choice of this equipment dictates the spatial resolution of
the calcium measurements, where large cell populations are used in cuvettes, and
microscopes allow single-cell and subcellular measurements. In recent years, ex-
citing methodology has emerged based on fluorescence microplate readers, a
development spurred on by the needs of pharmaceutical companies to efficiently
screen and identify bioactive molecules and therapeutic agents [1, 2]. As discussed
in Chapter 17, the ubiquitous calcium signal has taken a leading role in this respect,
with an array of advanced fluorescence microplate readers available that are
particularly suited for the high-throughput (HT) assessment of [Ca2þ]CYT. While a useful screening tool, the ability to measure changes in [Ca2þ]CYT is
key to help define and characterize underlying mechanisms of calcium signaling,
and the role of [Ca2þ]CYT in cellular events ranging from contraction to synaptic transmission to gene transcription [3]. However, the adoption of advanced micro-
plate readers for this purpose by basic research laboratories has been slow. High
capital equipment costs are likely a prohibitory factor, but these concerns should be
tempered by the advantages these systems can bring to the basic research environ-
ment. With increasing efficiency and flexibility for performing HT assessment of
[Ca2þ]CYT, the sophistication of HT-platforms to undertake complex cell signaling studies in HT mode continues to evolve. In this chapter, we will provide an
overview of the opportunities available for the HT assessment of [Ca2þ]CYT using microplate readers and some of the issues and challenges related to this technology.
Many aspects of the calcium signaling process can be described by directly obser-
ving [Ca2þ]CYT changes using fluorescent calcium indicators. Whether the equipment platform be cuvette-, microscope-, or microplate-based, it is critical to have a
fundamental understanding of the basis for measuring [Ca2þ]CYT levels. This includes appreciating the flexibility and limitations of applying fluorescent calcium
indicators. Chapter 2 outlines protocols that can be used to define underlying
calcium signaling processes, and minimize the potential for creating artifacts or
misinterpreting measurements.
