A versatile assay for the accurate, time-resolved determination of cellular viability

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Abstract

A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5×105 to 2.0×105 cells displayed a linear response when FDA concentrations less than 12 μM were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (Vmax) and the LD50 (half-maximal velocity or k1/2) were calculated as 17.2 (% death/h) and 65 nM, respectively.

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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