Preparation and use of sea urchin egg homogenates.
Morgan AJ., Galione A.
Cell homogenates provide a simple and yet powerful means of investigating the actions of Ca(2+)-mobilizing second messengers and their target Ca(2+) stores. The sea urchin egg homogenate is particularly useful and almost unique in retaining robust Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-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 Ca(2+) (or pHL) measurements, homogenate is thawed and sequentially diluted in an intracellular-like medium and the fluorescence of Ca(2+)- or pHL-sensitive dyes monitored in a standard fluorimeter or plate-reader.