Photolysis of caged calcium using a low-cost flash unit: efficacy analysis with a calcium selective electrode.
Photolysis of caged calcium (Nitr5, Calbiochem) can be used to study calcium dependent processes such as excitation-contraction coupling and muscular mechanics. Expensive high energy light sources are routinely used for UV light exposure, but this study describes an alternative low cost xenon flash unit constructed in our laboratory. A 300 J short arc xenon flash lamp (Heimann) was mounted in an elliptical reflector and driven by a modified Metz 60 CT 4 photoflash unit up to 240 J input energy and 4 ms flash duration. A 20 microliters cuvette containing a test solution was placed in a complementary elliptical reflector. An ion selective calcium electrode was used to measure the free calcium concentration [Ca2+] before and after flash in test solutions containing 1.00 mM Nitr5 in combination with different added [Ca2+]s. Using this technique we estimated that 1 flash on 1.00 mM Nitr5 increased the free [Ca2+] from 10(-7) to 1.1 x 10(-5) M. When the added [Ca2+] was less than 2.3 x 10(-4) M, the used Nitr5 behaved as a strong calcium chelator because 23% of it was unloaded with calcium. It is concluded that a physiologically relevant change in free [Ca2+] can be evoked by photolysis of Nitr5 using a low cost (approximately $1500) xenon flash unit, and that ion selective Ca electrodes can be adequately used to monitor the resulting changes in [Ca2+].
Department of Urology, Erasmus University Rotterdam, The Netherlands.
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Van Koeveringe
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Van Mastrigt
D002118:Calcium / Q000032:analysis*; D002614:Chelating Agents; D004533:Egtazic Acid / Q000031:analogs & derivatives; D017736:Ion-Selective Electrodes; D010782:Photolysis; D014466:Ultraviolet Rays
D002614:Chelating Agents; C052455:nitr 5; D004533:Egtazic Acid; D002118:Calcium