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Microtiter plate assay of yeast cell number using the fluorescent dye Calcofluor White M2R

2004; Future Science Ltd; Volume: 36; Issue: 3 Linguagem: Inglês

10.2144/04363bm01

1940-9818

Mateja Novak Štagoj, Radovan Komel, Aleksandra Comino,

Single-cell and spatial transcriptomics

BioTechniquesVol. 36, No. 3 BenchmarksOpen AccessMicrotiter plate assay of yeast cell number using the fluorescent dye Calcofluor White M2RMateja Novak Stagoj, Radovan Komel & Aleksandra CominoMateja Novak StagojNational Institute of Chemistry, Radovan KomelNational Institute of ChemistryInstitute of Biochemistry, University of Ljubljana, Ljubljana, Slovenia & Aleksandra Comino*Address correspondence to: Aleksandra Comino, National Institute of Chemistry, Laboratory for Biosynthesis and Biotransformation, Hajdrihova 19, SI-1000 Ljubljana, Slovenia. e-mail: E-mail Address: sasa.comino@ki.siNational Institute of ChemistryPublished Online:6 Jun 2018https://doi.org/10.2144/04363BM01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The development of a fast, simple, sensitive, and accurate method for counting cells in culture in various testing conditions is important for many fields of biology and biotechnology research. Several methods are widely used, each of which has advantages and limitations. One commonly used technique for counting yeast cells is measurement of optical density by spectrophotometry at an absorbance of 600 nm (A600). This method is simple and easy to handle, but the tendency of cells to settle down is often a limiting factor. Moreover, the narrow quantitation range necessitates laborious concentration adjustment that is unsuitable for high processivity of numerous culture samples with various cell densities. The estimation of cell density by cell counting in a heamocytometer is too laborious and time-consuming to be useful in high-throughput screening. The development of fluorescence indicators of cellular DNA content has made the process of quantitating cell number faster and more convenient. There are several dyes used in cell quantitation that exhibit fluorescence enhancement upon binding to DNA, such as Hoechst 33258 (1–4), Hoechst 33342 (5,6), DAPI (diaminophenylindole) (6,7), propidium iodide (8,9), and SYBR® Green I (10). Several methods have been described that quantitate cell number in the microtiter plate, although almost all require cumbersome and lengthy preparative steps or extraction of the bound dye from the cell or are not applicable to unfixed cells. A large number of assays employing quantitation of the cell number that rely on the cellular metabolic activity have also been developed (11,12); however, their utility is limited because the cellular metabolic processes vary over the time. Also, the method of quantification of dye metabolism is often incompatible with other assays.Here, we describe the correlation between A600 and fluorescence of Calcofluor White M2R dye binding to the cell surface of budding yeast. Fluorescent dye Calcofluor White M2R [also known as Fluorescent Brightener 28 (Sigma, St. Louis, MO, USA)] is frequently used in cell biology to stain plant cell walls and other cellulose-containing structures. In yeast, calcofluor binds to the chitin-rich bud scars present on a mother cell after cell division (13). Figure 1 presents spectral characteristics of bound calcofluor.Figure 1. Fluorescence excitation and emission spectra of Calcofluor White M2R bound to the yeast cell surface.Saccharomyces cerevisiaecells were stained with calcofluor using protocol described in the Table 1. The excitation spectrum of stained cells was recorded at an emission wavelength of 460 nm. The emission spectrum was recorded at an excitation wavelength of 360 nm.We developed and quantified a rapid, simple, fluorometric assay, which is readily adaptable to automation. The accuracy of the calcofluor binding was compared to A600 using a UV-Vis spectrophotometer (Hewlett-Packard, Palo Alto, CA, USA) (Figure 2). The correlation coefficient 0.99 is a result of linear regression of the emitted fluorescence as a function of A600, indicating that emitted fluorescence is directly proportional to the number of cells in the well.Figure 2. Correlation between A600and fluorescence of calcofluor binding to BY4741 cell surface at assay linearity at (A) high and (B) low cell counts.Experiments were done in triplicates. Data points are the mean (± sem) from three different experiments. Optical density was determined by absorbance measurement at 600 nm (A600). Fluorescence is given in arbitrary units (AU).Cell number determination can be easily performed according to the protocol shown in Table 1. Saccharomyces cerevisiae strains were cultured in sterile 96-well microtiter plates (Porvair Sciences, Shepperton, UK) in the standard media YEPD and YEPGal (BD Difco, Sparks, MD, USA) as previously described (14). Calcofluor White M2R stock solution was prepared by dissolving 48 mg solid dye in 10 mL water and filter sterilized. Stock solution can be stored at −20°C for up to 6 months. Calcofluor staining solution in buffer A was made fresh daily and maintained at room temperature in the dark. Unbound calcofluor emitted very low fluorescence, although better results were obtained if dye solution was removed before fluorescence reading. Therefore microplate cultures were harvested by centrifugation and gently inverted onto the paper towels to remove dye solution. Cells were resuspended in TE buffer [10 mM Tris-HCl, pH 8.0, 10 mM EDTA (Sigma)]. Florescence intensity was measured using a fluorescence spectrophotometer (LS55 microplate reader; Perkin Elmer, Wellesley, MA, USA) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm, respectively (2.5- and 5-nm slit widths). A 5-s reading was set for each microwell. Measurements were taken in triplicate and averaged. TE buffer was used as a blank standard. Fluorescence of unstained cells resuspended in TE buffer was negligible over the complete range of cell densities (almost the same as TE buffer). In each experiment, a standard calibration curve was generated by plotting fluorescence intensity versus A600 as determined previously by UVVis spectrophotometer.Table 1. Procedure for Fluorescence-Based Determination of Yeast Cell Number in Microtiter PlateUnder these conditions, curves were linear (r2 > 0.99), detecting as few as 1.5 × 105 cells per well and as many as 1.0 × 108 cells per well with a single dye concentration. Figure 2B shows that the slope is slightly lower at low cell concentration, and therefore the number of yeast cells is consequently slightly underestimated. The procedure is applicable to both unfixed and 4% formaldehyde/phosphate-buffered saline fixed cells. The suitability of the assay for different yeast strains was also tested. We measured different S. cerevisiae strains: BY4741 (Euroscarf, Frankfurt, Germany), CBL1-20 (15), and EGY48 (16). A high degree of correlation between A600 and emitted fluorescence was found for all strains. The fluorescence of chitin-dye complexes correlated linearly with the cell number over a broad range of all yeast strains tested. The assay is applicable for various yeast strains with different morphological characteristics. However, the standard curve needs to be determined for each yeast strain.Since calcofluor predominately binds to the bud scar (13), we measured cell population at various growth stages and found that quantitation was not dependent on the senescence of the culture. Standard curves of exponentially (logarithmic) growing cells and the stationary phase cells were nearly indistinguishable (data not shown), indicating that the assay can be applied to the cell number determination at various growth stages and metabolic conditions.One of the great advantages of the method is fluorescence stability. We exposed calcofluor-stained cells to UV light several times, and no or only very weak photobleaching was observed. Thus, several measurements of the same cells can be performed to obtain the same value.In this study, we evaluate calcofluor binding to the yeast cell wall and present feasibility of the resulting fluorescence for cell number determination in a 96-well format. The fluorescence-based method for cell number quantification we describe is especially appropriate when handling a number of samples with a wide range of cell densities. It offers a broad dynamic quantitation range relative to A600. This method could be used in conjunction with other fluorescence-based assays in microtiter plates either simultaneously or before/after the experiment, depending on the methodology of the assay and limitation of quantitation caused by light interference.In conclusion, high-throughput screening of cells based on fluorescence measurement of various parameters demands fast, reliable, and accurate determination of the cell number. Cell quantitation by calcofluor staining is an excellent alternative. It combines advantages of a fast, simple, and accurate technique that is low cost and reliable for a wide range of cell densities and various S. cerevisiae strains. 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Cell Biol. 15:5820–5829.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByHigh throughput comparative assessment of biofilm formation of Candida glabrata on polystyrene material11 February 2022 | Korean Journal of Chemical Engineering, Vol. 44Candida albicans biofilm formation and growth optimization for functional studies using response surface methodology25 January 2022 | Journal of Applied Microbiology, Vol. 247Heterologous expression of cyanobacterial gas vesicle proteins in Saccharomyces cerevisiae23 September 2021 | Biotechnology Journal, Vol. 16, No. 12Droplet Sensitized Fluorescence Detection for Enzyme-Linked Immune Sorbent Assays on Microwell Plate11 April 2019 | Analytical Chemistry, Vol. 91, No. 9Yeast: bridging the gap between phenotypic and biochemical assays for high-throughput screening16 October 2018 | Expert Opinion on Drug Discovery, Vol. 13, No. 12A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agentsLetters in Applied Microbiology, Vol. 44, No. 4Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiaeFEMS Microbiology Letters, Vol. 244, No. 1 Vol. 36, No. 3 Follow us on social media for the latest updates Metrics Downloaded 986 times History Received 1 December 2003 Accepted 7 January 2004 Published online 6 June 2018 Published in print March 2004 Information© 2004 Author(s)AcknowledgmentsThis work was supported by the Ministry of Education, Science and Sports of Slovenia, grant no. 3311-01-831711 to M. 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