Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader.
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 microg 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 x 10(6) cells to 10.0 x 10(6) 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.
Lamboursain
Laurence
L
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.
Jolicoeur
Mario
M
D006690:Bisbenzimidazole / Q000378:metabolism; D002452:Cell Count; D002478:Cells, Cultured; D018744:DNA, Plant / Q000378:metabolism; D031675:Eschscholzia / Q000166:cytology* / Q000254:growth & development; D005456:Fluorescent Dyes / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*
D018744:DNA, Plant; D005456:Fluorescent Dyes; D006690:Bisbenzimidazole