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.

Diverse Subcellular Locations of Cryptogein-Induced Reactive Oxygen Species Production in Tobacco Bright Yellow-2 Cells

“…For kinetic CLSM measurements of individual cells, the AR reagent proved to be less useful because it showed laser-induced instability (Zhou et al, 1997;Towne et al, 2004). As in the case of AR, the DCF signal is dependent on its concentration in a particular compartment and on the intrinsic cellular peroxidase activity (Gerber and Dubery, 2003). However, the kinetics of DCF oxidation within a certain compartment should represent actual real-time changes in H 2 O 2 concentrations that result from production in the compartment or from H 2 O 2 influx to the compartment.…”

In Vitro Inhibition of Streptococcus mutans Biofilm Formation on Hydroxyapatite by Subinhibitory Concentrations of Anthraquinones

“…In the first assay, MICs were determined in the presence or absence of 1.5 mM glutathione (GSH), 0.025% (wt/ vol) cysteine, and 10 mM mannitol (all from Sigma) by using the modified microdilution assay described above. In the second assay, we used the fluorescent probe 2Ј,7Ј-dichlorofluorescein diacetate (DCF-DA) to quantitate the amount of ROS produced (5,6). The inhibition of mutacin production can be used as an indirect assay to detect competence-stimulating peptide-based quorum sensing (QS) (22).…”

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

“…Most of the plant ROS visualization studies were otherwise performed on cell cultures [7,9]. The main drawback of these studies is dubious extrapolation of results to intact higher plants where the cells originated from.…”

“…Therefore, one could consider using the more expensive, carboxylated, form of H 2 DCFDA (carboxy-H 2 DCFDA) because of its better membrane permeability and better retention in the intracellular space [11]. According to our results, it could be argued that yeast cells [8] or plant cell cultures [7,9] are probably better test subjects than L. minor plants for intracellular ROS generation studies. This is primarily because of their big surface-to-volume ratio that enables faster dye uptake and thus short loading periods.…”

“…Dichlorofluorescin (H 2 DCF) is a fluorescent marker widely used for determination of H 2 O 2 [6,7], and for studies of oxidative stress at the cellular level [8,9]. In cellular studies, dichlorofluorescein (H 2 DCF) is given as a diacetate ester (dichlorofluorescein diacetate; H 2 DCFDA), which permeates the plasma membrane and is hydrolyzed inside the cells by unspecific esterases yielding polar products, supposedly trapped within the cell.…”