Measurement of intracellular Ca2+ concentration using Indo-1 during simultaneous flash photolysis to release Ca2+ from DM-nitrophen.
We have constructed a modular instrument to measure intracellular [Ca2+] ([Ca2+]i) in single isolated cells while simultaneously imposing step changes in [Ca2+]i using "caged Ca2+". By combining the outputs of a xenon arc lamp with a frequency-tripled (Nd:YAG) laser, the instrument can operate with low maintained illumination to measure [Ca2+]i using a ratiometric Ca(2+)-sensitive fluorophore and also activate the release of Ca2+ from a caged-Ca2+ compound with a high energy pulse of ultraviolet light. This instrument is simple to assemble, introduces little electrical noise, provides a wide range of illumination power, produces only moderate photobleaching of the Ca2+ indicator and can be readily adapted to diverse cellular preparations. We demonstrate the use of this system to measure step changes in [Ca2+]i in adult rat ventricular myocytes and a human embryonic kidney cell line (293 cells) in culture.
Kirby
M S
MS
Department of Physiology, University of Maryland at Baltimore 21201.
Hadley
R W
RW
Lederer
W J
WJ
D000085:Acetates / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D005029:Ethylenediamines / Q000378:metabolism*; D005456:Fluorescent Dyes; D006352:Heart Ventricles; D006801:Humans; D007211:Indoles; D007425:Intracellular Membranes / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007834:Lasers; D009200:Myocardial Contraction; D009206:Myocardium / Q000166:cytology; D009994:Osmolar Concentration; D010782:Photolysis; D051381:Rats; D014466:Ultraviolet Rays
D000085:Acetates; D002614:Chelating Agents; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D007211:Indoles; C057014:DM-nitrophen; C048960:indo-1; D002118:Calcium