A versatile assay for the accurate, time-resolved determination of cellular viability
Section snippets
Plant materials
Tobacco cell suspensions (BY-2) were maintained in 20 ml of MS medium containing sucrose (3%, w/v). Cultures were grown in Erlenmeyer flasks (100 ml) at 25 °C with shaking at 130 rpm. For FDA assays, cell suspensions were grown to saturation, which was reached 7 days after inoculation. An aliquot (7 ml) of this cell suspension was then used to inoculate fresh MS medium (100 ml) containing sucrose (3%, w/v) in a 300-ml Erlenmeyer flask. This culture was incubated at 25 °C for 4 days before use in the
In vivo quantification of cellular viability
In order to provide a simple means to accurately quantify the cell death-induction capabilities of different elicitors we have developed a time-resolved in vivo cell death assay.
First, to correctly calibrate the assay, we elaborated a set of viability standards using mixtures of living and dead tobacco BY-2 cells. The accuracy of these standards was established using two independent methods, Evans blue and propidium iodide assays, which allow the determination of quantities of living and dead
Conclusion
Here we have described a simple, efficient method for the accurate measurement of plant cell viability. Using cryptogein-induced cell death as a model, we have shown that this spectrofluorimetric FDA viability assay makes the quantification of the death response possible. Therefore, this method represents a new powerful tool for the detailed analysis of death initiation mechanisms in plant cells. Moreover, our data indicate that this assay should be applicable to a wide variety of plant cells.
Acknowledgements
This research was supported in part by grants from Grant-in Aid (No.12052211) to T.A. from the Ministry of Education, Shizuoka Scientific Organization, Hamamatsu Technoscience Organization, and the Sasagawa Scientific Research Grant from the Japan Science Society. Arabidopsis cultured cells were kindly donated by Dr. Masaaki Umeda and Dr. Yuji Moriyasu. Dr. Shinji Tsuyumu and Ms. Akiko Matsui are thanked for their useful suggestions throughout this work.
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Growth techniques
2023, The Chlamydomonas Sourcebook: Volume 1: Introduction to Chlamydomonas and Its Laboratory UseRemoval of field-collected Microcystis aeruginosa in pilot-scale by a jet pump cavitation reactor
2022, Ultrasonics SonochemistryCitation Excerpt :When the algal cells are treated by JPCR, the intracellular apparatus are greatly inhibited and damaged, leading to the loss of metabolic (esterase) activity and changes in cell membrane permeability. The treated algal samples are examined with an FDA reagent, and intact cells fluoresce green fluorescence when they are observed by fluorescence microscope at wavelength 493 nm, while almost dead cells do not fluoresce green due to the loss of metabolic (esterase) activity and changes in cell membrane permeability [8,51–53]. Fig. 6 shows the fluorescence microscope image of treated and untreated algal cells stained by the FDA reagent.
Effects of low-energy N<sup>+</sup>-beam implantation on root growth in Arabidopsis seedlings
2016, Ecotoxicology and Environmental SafetyCitation Excerpt :Furthermore, we found that the root tip length in the ion-implanted roots was significantly reduced compared with the length in control (Fig. 1E), implying that large doses of ion implantation suppressed the activities of cell division and elongation. The fluorescent dye FDA is well used as the indictor for cell viability (Amano et al., 2003). CyclinB1;1:GFP which represents a GFP fusion to an Arabidopsis mitotic cyclin can be used as a marker for cells about to enter, or in the process of, division (Moreno-Romero et al., 2008).
Grape marc extract causes early perception events, defence reactions and hypersensitive response in cultured tobacco cells
2014, Plant Physiology and BiochemistryCitation Excerpt :Primers and amplicon sizes are given in Appendix S1. The Evans Blue assay was performed according to Amano et al. (2003). Tobacco cells (500 μL) were withdrawn from the culture medium immediately after GME elicitation (t0) and then every 24 h for 3 days.
Hydrolysis and biomineralization of porous PLA microspheres and their influence on cell growth
2011, Colloids and Surfaces B: BiointerfacesCitation Excerpt :Cell viability was first evaluated by a rapid simultaneous staining method with FDA and PI as fluorescent dyes. Compared with other methods, this simultaneous staining method is more accurate, rapid and convenient [22,26,27]. The percentage of fluorescence intensity of living cells and dead cells (Iv/(Iv + Id)) has been reported to be in direct proportion to the percentage of the concentration of living cells and dead cells (Cv/(Cv + Cd)) (here Iv and Id represent the fluorescence intensity of living cells and dead cells; Cv and Cd represent the concentration of living cells and dead cells) [28].
Evaluation of power ultrasound for disinfection of both Legionella pneumophila and its environmental host Acanthamoeba castellanii
2010, Water ResearchCitation Excerpt :The concentration of infected A. castellanii and the mean amount of intracellular Legionella per infected host were determined as described previously by Declerck et al. (2005). Fluorescein diacetate (FDA) is widely used as an indicator of cell viability (Amano et al., 2003). Viable cells are capable to incorporate the nonpolar (thus cell permeable), nonfluorescent FDA compound and rapidly hydrolyze it using acetyl esterase activity to fluorescein, a polar fluorescent compound which is retained within the cell.