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Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader

  • Cell Biology and Morphogenesis
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Abstract

Microscopic counting of plant cells is a very tedious and time-consuming process and is therefore seldom used to evaluate plant cell number on a routine basis. This study describes a fast and simple method to evaluate cell concentration in a plant cell suspension using a fluorescence microplate reader. Eschscholtzia californica cells were fixed in a mix of methanol and acetic acid (3:1) and stained with a fluorescent DNA binding dye (Hoechst 33258). Readings were done in a fluorescence microplate reader at 360/465 nm. Specific binding of the dye to double-stranded DNA was significantly favored over unspecific binding when 1.0 M Tris buffer at pH 7.5 containing 1.0 M NaCl and 75 μg ml−1 of Hoechst 33258 was used. Fluorescence readings must be done between 4 min and 12 min following the addition of the staining solution to the sample. The microplate counting method provides a convenient, rapid and sensitive procedure for determining the cell concentration in plant cell suspensions. The assay has a linear detection range from 0.2×106 cells to 10.0×106 cells per milliliter (actual concentration in the tested cell suspension). The time needed to perform the microplate counting was 10% of that needed for the microscopic enumeration. However, this microplate counting method can only be used on genetically stable cell lines and on asynchronous cell suspensions.

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Acknowledgements

The authors would like to express their gratitude to the “Chaire CRSNG en bio-assainissement des sols”, who allowed the use of their microplate reader. We want also to thank Dr. Sylvain Mandeville for reviewing the manuscript. This research project was founded by The Natural Sciences and Engineering Research Council of Canada (NSERC) and the “Fonds pour la Formation de Chercheurs et l′Aide à la Recherche du Québec” (FQRNT)

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Authors and Affiliations

  1. Canada Research Chair on the Development of Metabolic Engineering Tools, Bio-P2 Research Unit, Department of Chemical Engineering, Ecole Polytechnique de Montréal, P.O. Box 6079, Station Centre-ville, Montreal, Quebec, Canada, H3C 3A7

    Laurence Lamboursain & Mario Jolicoeur

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  1. Laurence Lamboursain
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  2. Mario Jolicoeur
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Correspondence to Mario Jolicoeur.

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Communicated by L.C. Fowke

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Lamboursain, L., Jolicoeur, M. Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader. Plant Cell Rep 23, 665–672 (2005). https://doi.org/10.1007/s00299-004-0899-3

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  • DOI: https://doi.org/10.1007/s00299-004-0899-3

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