Abstract
Cell homogenates provide a simple and yet powerful means of investigating the actions of Ca2+-mobilizing second messengers and their target Ca2+ stores. The sea urchin egg homogenate is particularly useful and almost unique in retaining robust Ca2+ responses to all three major messengers, i.e., inositol 1,4,5-trisphosphate (IP3), cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate (NAADP) (Lee and Aarhus. J Biol Chem 270: 2152–2172, 1995). It is not only invaluable for probing the pharmacology and mechanism of action of these messengers, but can also be used to assay Ca2+ uptake mechanisms (Churchill et al. Cell 111: 703–708, 2002), second messenger production (Morgan et al. Methods in cADPR and NAADP research. In: Putney JW Jr (ed) Methods in calcium signalling, CRC: Boca Raton, FL, 2006), and dynamics of luminal pH (pHL) changes within acidic Ca2+ stores (Lee and Epel. Dev Biol 98: 446–454, 1983; Morgan and Galione. Biochem J 402: 301–310, 2007). Here, we detail the protocols for preparing and using egg homogenates, wherein eggs are shed and collected into artificial sea water (ASW), dejellied, washed several times in Ca2+-free ASW, and then finally washed and resuspended in an intracellular-like medium. Homogenization is effected with a Dounce glass tissue homogenizer (at 50 % (v/v)) and aliquots frozen and stored at −80 °C. For Ca2+ (or pHL) measurements, homogenate is thawed and sequentially diluted in an intracellular-like medium and the fluorescence of Ca2+- or pHL-sensitive dyes monitored in a standard fluorimeter or plate-reader.
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Acknowledgements
We thank the Wellcome Trust UK for funding and Clive Garnham and Lianne Davis for helpful discussions.
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Morgan, A.J., Galione, A. (2014). Preparation and Use of Sea Urchin Egg Homogenates. In: Carroll, D., Stricker, S. (eds) Developmental Biology of the Sea Urchin and Other Marine Invertebrates. Methods in Molecular Biology, vol 1128. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-974-1_10
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DOI: https://doi.org/10.1007/978-1-62703-974-1_10
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-974-1
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