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

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 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.

Critical influence of Eschscholzia californica cells nutritional state on secondary metabolite production

“…For example cell mass varied from 0.693 to 1.528 mg  10 À6 cells during the course of our experiments depending on cells nutritional status. For this reason cell concentration measurements are more reliable to assess plant cell proliferation in plant cell culture and can now be done very easily even with highly aggregated plant cells using a microplate reader (Lamboursain and Jolicoeur, 2004). It also seems crucial to use intracellular nutrient concentrations while developing models describing plant cells growth, as suggested by Curtis et al (1991) for suspension cells and Jolicoeur et al (2003) for hairy roots.…”

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

“…This measure is depicted in the upper part of figure 1. Figure 1 As reported in the literature [20,31,32,34,35], the diphenylamine and Hoechst 33258 ® based methods are only able to detect DNA, whereas the other tested protocols can also be used to quantify RNA.…”

“…As a blank 0.5ml EB-buffer was used. Different excitation wavelengths and emission wavelengths have been reported [20,31,32,34,35].…”

Transcriptional Changes in Damask Rose Suspension Cell Culture Revealed by RNA Sequencing

“…The rose cells were cultured continuously in a bioreactor, and 100 mL of the culture was sampled with the medium. After removing the medium using a nonwoven fabric, the sampled rose cells were weighed, and the cell count was determined using Laurence Lamboursain and Mario Jolicoeur’s method [ 21 ]. Cells with a mass of 1 mg were separated to the greatest extent possible and subjected to enzymatic treatment to facilitate the breakdown into smaller cellular entities, and the number of cells was measured using a hemocytometer.…”