DOES THE USE OF DM-NITROPHEN, NITR-5, OR DIAZO-2 INTERFERE WITH THE MEASUREMENT OF INDO-1 FLUORESCENCE

Emission spectra of the photolabile Ca2+ chelators DM-nitrophen, nitr- 5, and diazo-2 were studied alone, and in the presence of indo-1, to i nvestigate potential interactions that would make the simultaneous man ipulation and ratiometric measurement of the intracellular Ca2+ concen tration difficult. Neither diazo-2 nor its photoproduct were found to be significantly fluorescent, and consequently concentrations of diazo -2 up to 20 times that of indo-1 did not distort the emission spectra of indo-1. DM-nitrophen was scarcely fluorescent, but its fluorescence did increase upon photolysis. In contrast to diazo-2 and DM-nitrophen , nitr-5 itself was found to be quite fluorescent, and this fluorescen ce was significantly increased upon photolysis. Thus, combined use of nitr-5 and indo-1 poses the most difficulty. The emission spectra of a ll the investigated compounds were used to define experimental conditi ons and calibration procedures that make possible simultaneous measure ment and manipulation of the intracellular Ca2+ concentration.