Fluorescence microplate assay for the detection of oxidative burst products in tobacco cell suspensions using 2',7'-dichlorofluorescein.

Abstract

The technique of 2',7'-dihydrodichlorofluorescin diacetate (H2DCF-DA)-derived fluorescence was applied to measurements of the oxidative burst reaction in plant cell suspension cultures in an automatic fluorometric multiwell microplate assay. The developed procedure was found to be versatile and effective for the determination of the oxidative burst reaction in plant cell cultures. Using this assay, cumulative production of reactive oxygen intermediates may be monitored and recorded non-destructively on a real-time scale for a large number of samples at frequent and continual time intervals for time course experiments. Through the use of various inhibitors and inducers or elicitors of the oxidative burst in combination with H2DCF-DA, this assay aids in the dissection of the signal transduction pathways and the determination of the origins of the oxidative burst in plant cells.