Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks
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{"title"=>"Quantitative analysis of protein phosphorylations and interactions by Multi-Colour IP-FCM as an input for kinetic modelling of signalling networks", "type"=>"journal", "authors"=>[{"first_name"=>"Sumit", "last_name"=>"Deswal", "scopus_author_id"=>"23392031200"}, {"first_name"=>"Anna K.", "last_name"=>"Schulze", "scopus_author_id"=>"49061587300"}, {"first_name"=>"Thomas", "last_name"=>"Höfer", "scopus_author_id"=>"7006417468"}, {"first_name"=>"Wolfgang W A", "last_name"=>"Schamel", "scopus_author_id"=>"6602193405"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"362239072", "doi"=>"10.1371/journal.pone.0022928", "sgr"=>"79960915174", "scopus"=>"2-s2.0-79960915174", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"21829558"}, "id"=>"651dc214-d552-3337-ac2b-9a606f35edf3", "abstract"=>"BACKGROUND: To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success.\n\nMETHODOLOGY/PRINCIPAL FINDINGS: We use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation in the murine T-cell hybridoma and primary murine T cells. Counter-intuitively, these data showed that cell stimulation by pervanadate led to a transient decrease of the phospho-ZAP70/ZAP70 ratio at the TCR. A mechanistic mathematical model of the underlying processes demonstrated that an initial massive recruitment of non-phosphorylated ZAP70 was responsible for this behaviour. Further, the model predicted a temporal order of multisite phosphorylation of ZAP70 (with Y319 phosphorylation preceding phosphorylation at Y493) that we subsequently verified experimentally.\n\nCONCLUSIONS/SIGNIFICANCE: The quantitative data sets generated by IP-FCM are one order of magnitude more precise than Western blot data. This accuracy allowed us to gain unequalled insight into the dynamics of the TCR-CD3-ZAP70 signalling network.", "link"=>"http://www.mendeley.com/research/quantitative-analysis-protein-phosphorylations-interactions-multicolour-ipfcm-input-kinetic-modellin", "reader_count"=>36, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>12, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>7, "Other"=>1, "Student > Bachelor"=>1, "Professor"=>3}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>12, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>7, "Other"=>1, "Student > Bachelor"=>1, "Professor"=>3}, "reader_count_by_subject_area"=>{"Engineering"=>2, "Biochemistry, Genetics and Molecular Biology"=>3, "Mathematics"=>1, "Agricultural and Biological Sciences"=>23, "Medicine and Dentistry"=>1, "Business, Management and Accounting"=>2, "Physics and Astronomy"=>2, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>23}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Mathematics"=>{"Mathematics"=>1}}, "reader_count_by_country"=>{"Sweden"=>1, "Germany"=>2}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/751085"], "description"=>"<p>The model considers the phosphorylation of ITAMs by Lck, the binding of ZAP70 with its tandem SH2 domains to doubly phosphorylated ITAMs, the phosphorylation of Y319 of ZAP70 by Lck and of Y493 of ZAP70 by transphosphorylation. For the transphosphorylation two ZAP70 molecules both being in the opened conformation (phospho-Y319) have to be in close proximity. Therefore, the basic unit of the model is a pair of opposite ITAMs, as found in the paired CD3 and ζ chains. The scheme shows the possible configurations of the two ITAMs and bound ZAP70 molecules in the model, together with the reaction steps connecting the different states. Note that the two opposing ITAMs are treated as indistinguishable, so that it does not matter, for example, to which ITAM a ZAP70 molecule is bound. Likewise, the two phosphorylation sites within an ITAM are treated, for simplicity, as having identical properties. The differential equations governing the time evolution of the different states are given in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022928#s4\" target=\"_blank\">Materials and methods</a>.</p>", "links"=>[], "tags"=>["signalling", "events"], "article_id"=>421461, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g006", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mathematical_model_for_early_signalling_events_at_the_TCR_/421461", "title"=>"Mathematical model for early signalling events at the TCR.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:24:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/750839"], "description"=>"<p>(<b>a</b>, <b>b</b>) Splenocytes from OT-1 TCRαβ transgenic mice were stimulated with 5 mM pervanadate and lysed at different time points. The lysates were divided in two parts and used for three-colour IP-FCM as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022928#pone-0022928-g001\" target=\"_blank\">figure 1</a> (<b>a</b>) or for IP-WB measurements (<b>b</b>). In (<b>a</b>) the ratios of two MFIs after background subtraction from IP-FCM are shown. Error bars represent mean +/− s.e.m. (<b>c</b>, <b>d</b>) Splenocytes from OT-1 mice were stimulated with 100 nM OVA peptide-MHC tetramer ligand and lysed at different time points. The lysates were divided in two parts and used for three-colour IP-FCM as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022928#pone-0022928-g001\" target=\"_blank\">figure 1</a> (<b>c</b>) or for IP-WB measurements (<b>d</b>). In (<b>c</b>) the ratios of two MFIs after background subtraction from IP-FCM are shown. Error bars represent mean +/− s.e.m.</p>", "links"=>[], "tags"=>["ip-wb"], "article_id"=>421219, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_IP_FCM_and_IP_WB_measurement_using_primary_T_cells_/421219", "title"=>"IP-FCM and IP-WB measurement using primary T-cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:20:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/751018"], "description"=>"<p>(<b>a</b>) 2B4 T-cells were pervanadate-stimulated for 10 min and lysed. IP was done with anti-TCRβ coupled latex beads and the beads were individually stained with saturating conditions of anti-ZAP70-PE (red), anti-pY319-ZAP70-PE (orange) and anti-CD3ε-PE (blue) antibodies. PE-labelled Quantibrite beads (black) were measured along with the samples to generate a standard curve of MFI versus PE molecules per bead. (<b>b</b>) From the standard curve, PE molecules per bead for the IP-FCM samples were determined in a triplicate experiment. The number of ZAP70 molecules and pY319-ZAP70 molecules per TCR-CD3 and the number of pY319-ZAP70 per ZAP70 are shown. Mean ± s.e.m. values are displayed. (<b>c</b>) 2B4 cells were pervanadate-stimulated for 20 min and lysed. IP was done with anti-TCRβ coupled latex beads and the beads were stained either with anti-ZAP70-alexa488, anti-pY319-ZAP70-alexa647 and anti-CD3ε-APC antibodies together (simultaneous, three colour IP-FCM) or with one antibody at a time (individual). Histograms to compare these stainings are shown for each of the antibodies. The experiment was done in triplicate as indicated by the histogram colours.</p>", "links"=>[], "tags"=>["genetics and genomics", "Computational biology", "cell biology"], "article_id"=>421393, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_absolute_values_by_IP_FCM_/421393", "title"=>"Generation of absolute values by IP-FCM.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:23:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/751209"], "description"=>"<p>Least-square fitting of the differential equation system given in material and methods to the experimental data accurately reproduces the time courses of ZAP70 bound to the TCR-CD3 (<b>a</b>), pY319-ZAP70 bound to the TCR-CD3 and (<b>b</b>) the ratio of pY319-ZAP70 to ZAP70 both bound to CD3 (<b>c</b>). Data points are reproduced from <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022928#pone-0022928-g001\" target=\"_blank\">figure 1c</a>. The graphs are derived from the mathematical model.</p>", "links"=>[], "tags"=>["mathematical", "accounts", "experimentally", "observed", "kinetics", "zap70", "recruitment"], "article_id"=>421579, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g007", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_mathematical_model_accounts_for_the_experimentally_observed_kinetics_of_ZAP70_recruitment_and_phosphorylation_/421579", "title"=>"The mathematical model accounts for the experimentally observed kinetics of ZAP70 recruitment and phosphorylation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:26:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/750775"], "description"=>"<p>(<b>a</b>) Scheme of a two-plexed, two-colour IP-FCM quantifying the phosphorylation of CD3 and LAT. Anti-TCRβ- and anti-LAT-coupled beads are mixed for IP and distinguished in FCM according to their size. (<b>b</b>) 2B4 cells were stimulated as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022928#pone-0022928-g001\" target=\"_blank\">figure 1</a> and the two-plexed, two-colour IP-FCM was performed. The ratio of the MFIs of phospho-CD3ε and CD3ε and the MFI of phospho-LAT are shown. (<b>c</b>) The experiment was done as in (b) with the inclusion of 0.1 M phenyl-phosphate (PP) during the anti-phospho-tyrosine staining step. The MFI of phosphorylated CD3 at 10 min is shown. Mean ± s.e.m. values are displayed.</p>", "links"=>[], "tags"=>["genetics and genomics", "Computational biology", "cell biology"], "article_id"=>421148, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expansion_of_IP_FCM_/421148", "title"=>"Expansion of IP-FCM.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:19:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/750901"], "description"=>"<p>2B4 cells were stimulated with anti-CD3 antibodies for different time points or with pervanadate for 5 min. Two-colour IP-FCM purifying Erk and staining with anti-Erk-alexa488 and anti-phospho-Erk-alexa647 was done. Dot plots (<b>a</b>) and the ratio (<b>b</b>) of the MFIs are shown. The control in (a) is as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022928#pone-0022928-g001\" target=\"_blank\">figure 1b</a>. (<b>c</b>) Lysates from (a) were separated on a 12% SDS-PAGE. The WB membrane was developed using anti-phospho-Erk and anti-GAPDH antibodies as a loading control. Signal intensities were determined using the chemo-luminescence detection system. (<b>d</b>) The phospho-Erk signal intensity was normalized with respect to that of GAPDH.</p>", "links"=>[], "tags"=>["genetics and genomics", "Computational biology", "cell biology"], "article_id"=>421273, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantification_of_phospho_Erk_/421273", "title"=>"Quantification of phospho-Erk.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:21:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/378024", "https://ndownloader.figshare.com/files/378051", "https://ndownloader.figshare.com/files/378072", "https://ndownloader.figshare.com/files/378091", "https://ndownloader.figshare.com/files/378110", "https://ndownloader.figshare.com/files/378125", "https://ndownloader.figshare.com/files/378158", "https://ndownloader.figshare.com/files/378199", "https://ndownloader.figshare.com/files/378241", "https://ndownloader.figshare.com/files/378278", "https://ndownloader.figshare.com/files/378317"], "description"=>"<div><h3>Background</h3><p>To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success.</p> <h3>Methodology/Principal Findings</h3><p>We use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation in the murine T-cell hybridoma and primary murine T cells. Counter-intuitively, these data showed that cell stimulation by pervanadate led to a transient decrease of the phospho-ZAP70/ZAP70 ratio at the TCR. A mechanistic mathematical model of the underlying processes demonstrated that an initial massive recruitment of non-phosphorylated ZAP70 was responsible for this behaviour. Further, the model predicted a temporal order of multisite phosphorylation of ZAP70 (with Y319 phosphorylation preceding phosphorylation at Y493) that we subsequently verified experimentally.</p> <h3>Conclusions/Significance</h3><p>The quantitative data sets generated by IP-FCM are one order of magnitude more precise than Western blot data. This accuracy allowed us to gain unequalled insight into the dynamics of the TCR-CD3-ZAP70 signalling network.</p> </div>", "links"=>[], "tags"=>["quantitative", "phosphorylations", "interactions", "multi-colour", "ip-fcm", "kinetic", "signalling", "networks"], "article_id"=>134707, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0022928.s001", "https://dx.doi.org/10.1371/journal.pone.0022928.s002", "https://dx.doi.org/10.1371/journal.pone.0022928.s003", "https://dx.doi.org/10.1371/journal.pone.0022928.s004", "https://dx.doi.org/10.1371/journal.pone.0022928.s005", "https://dx.doi.org/10.1371/journal.pone.0022928.s006", "https://dx.doi.org/10.1371/journal.pone.0022928.s007", "https://dx.doi.org/10.1371/journal.pone.0022928.s008", "https://dx.doi.org/10.1371/journal.pone.0022928.s009", "https://dx.doi.org/10.1371/journal.pone.0022928.s010", "https://dx.doi.org/10.1371/journal.pone.0022928.s011"], "stats"=>{"downloads"=>3, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Quantitative_Analysis_of_Protein_Phosphorylations_and_Interactions_by_Multi_Colour_IP_FCM_as_an_Input_for_Kinetic_Modelling_of_Signalling_Networks/134707", "title"=>"Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-07-29 01:18:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/751260"], "description"=>"<p>(<b>a</b>) An experiment as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022928#pone-0022928-g001\" target=\"_blank\">figure 1</a> was performed, including a 60 min stimulation, in which pervanadate was applied at time 0 and the stimulation was stopped at time 60. A second 60 min stimulation was done, in which pervanadate was applied at time 0 and again at time 20 and the stimulation was stopped at time 60 (60+40). (<b>b</b>) Unstimulated 2B4 T-cells were lysed and TCR-CD3 complexes or total ZAP70 were immunoprecipitated using anti-TCRβ- or anti-ZAP70-coupled latex beads. Beads were stained with anti-pY319-ZAP70-PE and anti-ZAP70-alexa488 antibodies and measured by FCM as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022928#pone-0022928-g001\" target=\"_blank\">figure 1</a>. The ratio of pY319-ZAP70 to ZAP70 is displayed**: p<0.05. Mean ± s.e.m. values are displayed.</p>", "links"=>[], "tags"=>["genetics and genomics", "Computational biology", "cell biology"], "article_id"=>421636, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g008", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Model_validation_/421636", "title"=>"Model validation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:27:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/751312"], "description"=>"<p>(<b>a</b>) Using the model, the phosphorylation kinetics of ZAP70 at Y319 and Y493 was simulated and plotted. Phosphorylation at Y493 was delayed with respect to Y319. (<b>b</b>) 2B4 T-cells were stimulated with 5 mM pervanadate for the indicated time points. After lysis the TCR-CD3 was immuno-precipitated and proteins subjected to SDS-PAGE and WB using anti-pY493-ZAP70 and anti-CD3ζ antibodies. The experiment was done in triplicates and band intensities were determined. The ratio of pY493-ZAP70 to CD3ζ is shown in arbitrary units. Mean ± s.e.m. values are shown.</p>", "links"=>[], "tags"=>["phosphorylation"], "article_id"=>421689, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g009", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ZAP70_phosphorylation_at_Y493_/421689", "title"=>"ZAP70 phosphorylation at Y493.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:28:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/750644"], "description"=>"<p>(<b>a</b>) Anti-TCRβ-coupled latex beads are used for immuno-precipitation of the TCR-CD3 complex from cell lysates. Purified proteins are stained with flourophore-conjugated antibodies recognizing CD3ε, ZAP70 and phosphorylated ZAP70 at position Y319. Fluorescence of the beads is measured by FCM. (<b>b</b>) Murine 2B4 T-cells were stimulated with 5 mM pervanadate for the indicated time points. After lysis three-colour IP-FCM was performed. Dot plots of CD3ε, ZAP70 and pY319-ZAP70 intensities are shown. As a staining control (contr), uncoupled beads were used. (<b>c</b>) The experiment from (b) was done in triplicates and the ratios of two geometric mean fluorescence intensities (MFI) after background subtraction are shown in the arbitrary units. (<b>d</b>) Cells were stimulated as in (b), the TCR-CD3 was immuno-precipitated and proteins subjected to SDS-PAGE and WB using the antibodies indicated. (<b>e</b>) The experiment from (d) was done in triplicates and band intensities were determined. The ratios of two intensities are shown. Mean ± s.e.m. values are graphed in (c) and (e).</p>", "links"=>[], "tags"=>["genetics and genomics", "Computational biology", "cell biology"], "article_id"=>421017, "categories"=>["Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Sumit Deswal", "Anna K. Schulze", "Thomas Höfer", "Wolfgang W. A. Schamel"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022928.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Three_colour_IP_FCM_/421017", "title"=>"Three-colour IP-FCM.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-29 00:16:57"}

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