A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1
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{"title"=>"A simple FCM method to avoid misinterpretation in Saccharomyces cerevisiae cell cycle assessment between G0 and sub-G1", "type"=>"journal", "authors"=>[{"first_name"=>"Pierre", "last_name"=>"Delobel", "scopus_author_id"=>"55899531800"}, {"first_name"=>"Catherine", "last_name"=>"Tesnière", "scopus_author_id"=>"55969513800"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"372426320", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0084645", "scopus"=>"2-s2.0-84894255675", "pmid"=>"24392149", "sgr"=>"84894255675"}, "id"=>"6d744dd0-4126-3085-b65d-91cb820b22dd", "abstract"=>"Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of Saccharomyces cerevisiae cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application.", "link"=>"http://www.mendeley.com/research/simple-fcm-method-avoid-misinterpretation-saccharomyces-cerevisiae-cell-cycle-assessment-between-g0", "reader_count"=>53, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>1, "Researcher"=>14, "Student > Ph. D. Student"=>24, "Student > Master"=>2, "Student > Bachelor"=>8, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>1, "Researcher"=>14, "Student > Ph. D. Student"=>24, "Student > Master"=>2, "Student > Bachelor"=>8, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Engineering"=>2, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>32, "Medicine and Dentistry"=>3, "Chemical Engineering"=>1, "Chemistry"=>3, "Computer Science"=>1, "Earth and Planetary Sciences"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Chemistry"=>{"Chemistry"=>3}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>32}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>1}, "Chemical Engineering"=>{"Chemical Engineering"=>1}}, "reader_count_by_country"=>{"United States"=>2, "Denmark"=>1, "France"=>1, "Thailand"=>1, "Spain"=>1, "India"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1337831"], "description"=>"<p>Arrows underline the main evolutions of the DNA content from the lag phase to exponential growth (A, C, E) and from exponential growth to the stationary phase (B, D, F). Colors correspond to the different stages as defined in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084645#pone-0084645-g002\" target=\"_blank\">Figure 2</a>.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "microbiology", "Mycology", "yeast", "Applied microbiology", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "cytometry", "flow cytometry", "dimensional", "revealed", "sytox", "sybr"], "article_id"=>893389, "categories"=>["Biological Sciences"], "users"=>["Pierre Delobel", "Catherine Tesnière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0084645.g005", "stats"=>{"downloads"=>1, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_One_dimensional_representation_of_the_apparent_cell_size_A_B_and_DNA_content_as_revealed_by_SYTOX_Green_C_D_and_SYBR_Green_I_E_F_labeling_/893389", "title"=>"One dimensional representation of the apparent cell size (A, B), and DNA content as revealed by SYTOX Green (C, D) and SYBR Green I (E, F) labeling.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-02 04:11:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1337827"], "description"=>"<p>Five growth stages were defined according to log OD of the cell culture: 1: lag phase (black), 2: start of growth (cyan), 3: exponential stage (green), 4: slowdown phase (orange), 5: stationary phase (red).</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "microbiology", "Mycology", "yeast", "Applied microbiology", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "cytometry", "flow cytometry", "stages"], "article_id"=>893385, "categories"=>["Biological Sciences"], "users"=>["Pierre Delobel", "Catherine Tesnière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0084645.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Evolution_of_cell_density_and_identification_of_growth_stages_before_DNA_staining_/893385", "title"=>"Evolution of cell density and identification of growth stages before DNA staining.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-02 04:11:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1337826"], "description"=>"<p>Budding yeast cell progression through the cell cycle.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "microbiology", "Mycology", "yeast", "Applied microbiology", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "cytometry", "flow cytometry", "progression"], "article_id"=>893384, "categories"=>["Biological Sciences"], "users"=>["Pierre Delobel", "Catherine Tesnière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0084645.g001", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Budding_yeast_cell_progression_through_the_cell_cycle_/893384", "title"=>"Budding yeast cell progression through the cell cycle.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-02 04:11:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1337835"], "description"=>"<p>Global signal of low quality and upper G0 fluorescence.</p><p><sup>y</sup> presence of a sub-G1 peak out of the exponential growth.</p><p><sup>n</sup> absence of a sub-G1 peak out of the exponential growth.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "microbiology", "Mycology", "yeast", "Applied microbiology", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "cytometry", "flow cytometry", "fluorochromes", "exponential", "stationary"], "article_id"=>893393, "categories"=>["Biological Sciences"], "users"=>["Pierre Delobel", "Catherine Tesnière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0084645.t001", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Signal_intensity_with_different_fluorochromes_during_the_exponential_cell_growth_phase_G1_and_G2_M_or_stationary_phase_G0_/893393", "title"=>"Signal intensity with different fluorochromes during the exponential cell growth phase (G1 and G2/M) or stationary phase (G0).", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-01-02 04:11:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1337834"], "description"=>"<p>Gating was optimized for the whole growth experiment, and is summarized by the representation of yeast populations at three typical stages: exponential (245 <i>min</i>), slowdown (485 <i>min</i>) and stationary (725 <i>min</i></p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "microbiology", "Mycology", "yeast", "Applied microbiology", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "cytometry", "flow cytometry", "dimensional", "staining", "gating", "fluorochromes", "7-aad"], "article_id"=>893392, "categories"=>["Biological Sciences"], "users"=>["Pierre Delobel", "Catherine Tesnière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0084645.g006", "stats"=>{"downloads"=>2, "page_views"=>62, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Two_dimensional_representation_DNA_staining_signal_apparent_size_used_to_determine_gating_parameters_with_different_fluorochromes_SYTOX_174_Green_PI_TO_PRO_174_3_7_AAD_and_SYBR_174_Green_I_/893392", "title"=>"Two dimensional representation (DNA staining signal/apparent size) used to determine gating parameters with different fluorochromes (SYTOX® Green, PI, TO-PRO®-3, 7-AAD and SYBR® Green I).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-02 04:11:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1337829"], "description"=>"<p>The x axes are in arbitrary units. The y axes correspond to relative frequencies adjusted for each signal. Each histogram represents about 20000 <i>cells</i> Colors correspond to the different growth stages as defined in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084645#pone-0084645-g002\" target=\"_blank\">Figure 2</a>. For B to F, the vertical lines indicates the median signal (coefficient of variation) corresponding strictly to yeast cells at G1 stage during the exponential phase.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "microbiology", "Mycology", "yeast", "Applied microbiology", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "cytometry", "flow cytometry", "cytometric", "propidium", "iodide", "7-aminoactinomycin"], "article_id"=>893387, "categories"=>["Biological Sciences"], "users"=>["Pierre Delobel", "Catherine Tesnière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0084645.g004", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Evolution_of_the_cytometric_signal_during_cell_culture_time_in_min_with_A_cell_size_Forward_SCatter_B_SYTOX_174_Green_C_propidium_iodide_PI_D_TO_PRO_3_E_7_aminoactinomycin_D_7_AAD_and_F_SYBR_174_Green_I_/893387", "title"=>"Evolution of the cytometric signal during cell culture (time in min) with A: cell size (Forward SCatter), B: SYTOX® Green, C: propidium iodide (PI), D: TO-PRO-3, E: 7-aminoactinomycin D (7-AAD) and F: SYBR® Green I.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-02 04:11:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1337828"], "description"=>"<p>Colored scale indicates the stages as defined in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084645#pone-0084645-g002\" target=\"_blank\">Figure 2</a>.</p>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "microbiology", "Mycology", "yeast", "Applied microbiology", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "cytometry", "flow cytometry", "courses", "proportions", "populations", "fluorochrome"], "article_id"=>893386, "categories"=>["Biological Sciences"], "users"=>["Pierre Delobel", "Catherine Tesnière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0084645.g003", "stats"=>{"downloads"=>2, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Time_courses_of_the_relative_proportions_of_the_cell_populations_during_the_various_cell_cycle_stages_using_SYTOX_174_Green_fluorochrome_G0_black_G1_purple_S_green_and_G2_M_magenta_/893386", "title"=>"Time courses of the relative proportions of the cell populations during the various cell cycle stages, using SYTOX® Green fluorochrome (G0: black; G1: purple; S: green and G2/M: magenta).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-02 04:11:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/1337836"], "description"=>"<div><p>Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of <i>Saccharomyces cerevisiae</i> cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application.</p></div>", "links"=>[], "tags"=>["Biochemistry", "Nucleic acids", "dna", "microbiology", "Mycology", "yeast", "Applied microbiology", "Model organisms", "Yeast and fungal models", "Saccharomyces cerevisiae", "Molecular cell biology", "cytometry", "flow cytometry", "fcm", "misinterpretation", "g0", "sub-g1"], "article_id"=>893394, "categories"=>["Biological Sciences"], "users"=>["Pierre Delobel", "Catherine Tesnière"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0084645", "stats"=>{"downloads"=>7, "page_views"=>51, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Simple_FCM_Method_to_Avoid_Misinterpretation_in_Saccharomyces_cerevisiae_Cell_Cycle_Assessment_between_G0_and_Sub_G1/893394", "title"=>"A Simple FCM Method to Avoid Misinterpretation in <i>Saccharomyces cerevisiae</i> Cell Cycle Assessment between G0 and Sub-G1", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-02 04:11:04"}

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Relative Metric

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