Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays
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{"title"=>"Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays", "type"=>"journal", "authors"=>[{"first_name"=>"Ridha", "last_name"=>"Limame", "scopus_author_id"=>"35300053600"}, {"first_name"=>"An", "last_name"=>"Wouters", "scopus_author_id"=>"16833887800"}, {"first_name"=>"Bea", "last_name"=>"Pauwels", "scopus_author_id"=>"7003999875"}, {"first_name"=>"Erik", "last_name"=>"Fransen", "scopus_author_id"=>"7004158064"}, {"first_name"=>"Marc", "last_name"=>"Peeters", "scopus_author_id"=>"23012760000"}, {"first_name"=>"Filip", "last_name"=>"Lardon", "scopus_author_id"=>"6603745277"}, {"first_name"=>"Olivier", "last_name"=>"de Wever", "scopus_author_id"=>"15828854900"}, {"first_name"=>"Patrick", "last_name"=>"Pauwels", "scopus_author_id"=>"56975439800"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"365883747", "arxiv"=>"arXiv:1011.1669v3", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0046536", "scopus"=>"2-s2.0-84867676788", "pmid"=>"23094027", "sgr"=>"84867676788"}, "id"=>"4840fe0a-3417-30d6-9a58-492caa7ee559", "abstract"=>"BACKGROUND Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0-100 nM) correlated well with SRB (Rho>0.95) with similar IC(50) values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. CONCLUSIONS/SIGNIFICANCE The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance.", "link"=>"http://www.mendeley.com/research/comparative-analysis-dynamic-cell-viability-migration-invasion-assessments-novel-realtime-technology", "reader_count"=>179, "reader_count_by_academic_status"=>{"Unspecified"=>7, "Professor > Associate Professor"=>6, "Researcher"=>40, "Student > Doctoral Student"=>7, "Student > Ph. D. 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Student"=>53, "Student > Postgraduate"=>7, "Student > Master"=>34, "Other"=>3, "Student > Bachelor"=>14, "Lecturer"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>5}, "reader_count_by_subject_area"=>{"Unspecified"=>11, "Agricultural and Biological Sciences"=>86, "Business, Management and Accounting"=>1, "Chemistry"=>10, "Computer Science"=>2, "Engineering"=>10, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>22, "Nursing and Health Professions"=>3, "Materials Science"=>1, "Medicine and Dentistry"=>20, "Neuroscience"=>2, "Pharmacology, Toxicology and Pharmaceutical Science"=>5, "Physics and Astronomy"=>4, "Psychology"=>1}, "reader_count_by_subdiscipline"=>{"Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>20}, "Physics and Astronomy"=>{"Physics and Astronomy"=>4}, "Psychology"=>{"Psychology"=>1}, "Unspecified"=>{"Unspecified"=>11}, "Environmental Science"=>{"Environmental Science"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>5}, "Engineering"=>{"Engineering"=>10}, "Chemistry"=>{"Chemistry"=>10}, "Neuroscience"=>{"Neuroscience"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>86}, "Computer Science"=>{"Computer Science"=>2}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>3}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>22}}, "reader_count_by_country"=>{"Sweden"=>1, "United States"=>2, "Ireland"=>1, "Denmark"=>1, "Italy"=>1, "United Kingdom"=>1, "South Africa"=>1, "Australia"=>1, "Germany"=>2}, "group_count"=>7}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/556056"], "description"=>"*<p>Matrigel (Basement Membrane Matrix, growth factor reduced, BD Biosciences) delivered as a ±13.55 µg/µL stock.</p>", "links"=>[], "tags"=>["densities", "corresponding", "dilution", "20"], "article_id"=>226547, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536.t001", "stats"=>{"downloads"=>7, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Matrigel_surface_densities_corresponding_with_degree_of_dilution_for_a_fixed_volume_of_20_181_L_/226547", "title"=>"Matrigel surface densities corresponding with degree of dilution for a fixed volume of 20 µL.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-10-19 01:49:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/555973"], "description"=>"<p>A. Schematic depiction of processing kinetic data generated by SRB, <i>xCELLigence</i> and Transwells. Raw data with high time resolution (<i>filled</i> and <i>empty circles</i>), resulting from independent <i>xCELLigence</i> experiments (1, 2, 3 and <i>grey arrows</i>) are reduced to a lower time resolution by selecting only the data points corresponding with the time points of endpoint detection (<i>filled circles</i> only). Subsequently, data have been normalized by dividing all values by the highest value recorded over all experiments per method, resulting in a modified Y-axis scale that ranges from 0 to 1. Finally, the normalized data have been averaged with calculation of SD for the three independent experiments per method. B. Reduction of high-resolution data, generated by <i>xCELLigence</i>, to a low resolution comparable with data from conventional assays. The example shows migration (left) and invasion (<i>right</i>) of MDA-MB-231 cells through two densities of Matrigel. The ten time points in the Transwell method (<i>black arrows</i>) were selected from the <i>xCELLigence</i> plots (<i>grey</i> and <i>blue</i>) to reconstruct a low-resolution graph (<i>black</i>), directly comparable to the Transwell data. An identical approach was applied for all other processes studied.</p>", "links"=>[], "tags"=>["cell biology", "oncology", "biophysics", "developmental biology", "pathology"], "article_id"=>226458, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536.g007", "stats"=>{"downloads"=>4, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Prestatistical_data_processing_/226458", "title"=>"Prestatistical data processing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-10-19 01:47:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/296226"], "description"=>"<div><h3>Background</h3><p>Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based <em>xCELLigence</em> Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions.</p> <h3>Methodology/Principal Findings</h3><p>Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using <em>xCELLigence</em> technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0–100 nM) correlated well with SRB (Rho>0.95) with similar IC<sub>50</sub> values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method.</p> <h3>Conclusions/Significance</h3><p>The <em>xCELLigence</em> RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance.</p> </div>", "links"=>[], "tags"=>["comparative", "assessments", "Real-time", "endpoint", "assays"], "article_id"=>118357, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536", "stats"=>{"downloads"=>3, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Comparative_Analysis_of_Dynamic_Cell_Viability_Migration_and_Invasion_Assessments_by_Novel_Real_Time_Technology_and_Classic_Endpoint_Assays/118357", "title"=>"Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-10-19 02:19:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/555786"], "description"=>"<p>Comparison of random migration signals (negative control – SF) between three quantitation methods: pixel area calculation – <i>black</i>, OD - <i>red</i>, xCELLigence - <i>green</i>. A likelihood ratio test revealed a significant difference in slope between area calculation and OD (p<0.001) and area calculation and <i>xCELLigence</i> (p<0.001) for both cell lines and OD and <i>xCELLigence</i> (p<0.001) for MDA-MB-231 only. OD and <i>xCELLigence</i> slopes did not differ significantly (p = 0.22) for A549 cells.</p>", "links"=>[], "tags"=>["mda-mb-231"], "article_id"=>226278, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536.g005", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Time_dependent_random_migration_profile_of_MDA_MB_231_and_A549_/226278", "title"=>"Time-dependent random migration profile of MDA-MB-231 and A549.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-10-19 01:44:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/555386"], "description"=>"<p>Interdigitated gold microelectrodes on the well bottom (viability – E-plate) or on the bottom side of a filter membrane (motility – CIM-plate 16) detect impedance changes, caused by the presence of cells and expressed as a Cell Index. This detection method is proportional to both cell number (<i>left</i> and <i>above</i>) and morphology as increased cell spreading is reflected by a higher Cell Index value (<i>right</i>). When starting an experiment, a baseline Cell Index value is recorded in medium only before cell addition.</p>", "links"=>[], "tags"=>["impedance-based", "detection", "viability"], "article_id"=>225872, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536.g001", "stats"=>{"downloads"=>5, "page_views"=>132, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_xCELLigence_RTCA_impedance_based_detection_of_cell_viability_and_motility_/225872", "title"=>"<i>xCELLigence</i> RTCA: impedance-based detection of cell viability and motility.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-10-19 01:37:52"}
  • {"files"=>["https://ndownloader.figshare.com/files/555457"], "description"=>"<p>A. Proliferation curves of MDA-MB-231 cells as generated by <i>xCELLigence</i> RTCA (<i>red</i>) and SRB (<i>black</i>) for different seeding densities of 100 (<i>top left</i>), 500 (<i>bottom left</i>), 1000 (<i>top right</i>) and 2000 cells/cm<sup>2</sup> (<i>bottom right</i>) during a ten-day incubation. B. Same as (A) for A549 cells. All graphs represent results from three independent experiments ± SD. C. Cytotoxicity profiles relating to 72 hours of exposure to paclitaxel (0–100 nM). Cells were allowed to attach and propagate during 24 hours prior to start of treatment. Toxicity data from <i>xCELLigence</i> RTCA were derived from normalized plots. All graphs represent results from three independent experiments ± SD.</p>", "links"=>[], "tags"=>["proliferation", "cytotoxicity", "profiles", "mda-mb-231"], "article_id"=>225949, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536.g002", "stats"=>{"downloads"=>2, "page_views"=>60, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Time_dependent_proliferation_and_cytotoxicity_profiles_of_MDA_MB_231_and_A549_/225949", "title"=>"Time-dependent proliferation and cytotoxicity profiles of MDA-MB-231 and A549.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-10-19 01:39:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/555552"], "description"=>"<p>A. A Transwell setup consists of an upper chamber (<i>insert</i>) that is placed onto a lower chamber (<i>well</i>). The insert contains a microporous membrane (8 µm pores) allowing passage of tumor cells. After a period of serum starvation a serum-free cell suspension is seeded in the insert and exposed to medium containing potential chemoattractants (by default: medium+FBS). During incubation at 37°C and 5% CO<sub>2</sub>, cells migrate toward the bottom side of the membrane. B. Experimental design to assess time-dependent migratory behavior of cultured cells. Both migration toward FBS-containing medium and baseline migration (toward SF medium, no chemoattraction) as a negative control were included. Two times two 24-well Transwell plates were used to examine migration to FBS (positive control – <i>top row</i>) and baseline migration (negative control – <i>bottom row</i>). At ten time points during a 24-hour incubation period inserts were fixed and stained in duplicate. Two inserts containing cell-free media (grey fill) have been included throughout the experiment and fixed and stained after 12 hours incubation to assess background absorption in optical density (OD) measurements. In addition, to exclude influence of inter-plate variability on observed migration rates, each plate contained duplicate two-hour control inserts.</p>", "links"=>[], "tags"=>["transwell", "detection", "time-dependent"], "article_id"=>226041, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536.g003", "stats"=>{"downloads"=>2, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Conventional_Transwell_design_for_detection_of_time_dependent_cell_migration_/226041", "title"=>"Conventional Transwell design for detection of time-dependent cell migration.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-10-19 01:40:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/555873"], "description"=>"<p>A. Experimental design to quantify MDA-MB-231 Matrigel invasion. Two times two 24-well Transwell plates were used to examine invasion to FBS through a 20% (v/v) (<i>top row</i>) and 7.7% (v/v) Matrigel layer (<i>bottom row</i>) after 24 hours of serum starvation. At ten time points during a 48-hour incubation period inserts were fixed and stained in duplicate. Two inserts containing cell-free media (grey fill) have been included throughout the experiment and fixed and stained after 24 hours incubation to assess background absorption in optical density (OD) measurements. In addition, to exclude influence of inter-plate variability on observed migration rates, each plate contained duplicate 24-hour control inserts. B. MDA-MB-231 dynamic cell invasion profiles, generated by Transwell experiments (<i>black</i>) and <i>xCELLigence</i> (<i>red</i>). Graphs represent normalized signals (scaled values 0–1) of invasion through 20% (<i>open circles</i>), 10% (<i>open squares</i>), 7.7% (<i>filled circles</i>) and 3.3% (<i>filled squares</i>) to medium+10% FBS with associated Spearman's rank correlation coefficients (Rho). All results are from three independent duplicate experiments with SD. C. Sequential pictures showing invasive MDA-MB-231 cells at the indicated time points during a 48-hour incubation on Transwells coated with 20% (<i>top row</i>) and 7.7% Matrigel (<i>bottom row</i>). Pictures (obj. 2.5×) show cells fixed and stained in 20% methanol/0.1% crystal violet.</p>", "links"=>[], "tags"=>["cell biology", "oncology", "biophysics", "developmental biology", "pathology"], "article_id"=>226360, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536.g006", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Time_dependent_invasion_profile_of_MDA_MB_231_/226360", "title"=>"Time-dependent invasion profile of MDA-MB-231.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-10-19 01:46:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/555670"], "description"=>"<p>A. MDA-MB-231 (<i>left</i>) and A549 (<i>right</i>) cell migration profiles, detected by Transwell experiments (<i>black</i>) and <i>xCELLigence</i> (<i>red</i>). Graphs represent scaled signals (0–1) of net chemoattraction after subtraction of the random migration signal (<i>empty squares</i> in panel A, B), with associated Spearman's Rho values. All results originate from three independent duplicate experiments ± SD. B. Normalization procedure of migration patterns. Raw data (<i>left panel</i>) were normalized to a (0–1) scale (<i>middle panel</i>) through division of all data by the maximum value obtained in three independent experiments. Subsequently, random migration (SF) signals (<i>triangle markers</i>) were subtracted from the positive (FBS) control counterparts (<i>circle markers</i>) per experiment to obtain a pure chemotactic signal (<i>right panel</i>). Example shown is the migratory pattern of MDA-MB-231 cells estimated by pixel area calculation in three experiments (exp 1 - <i>red</i>, exp 2 - <i>green</i>, exp 3 - <i>black</i>). Triangle and circle markers represent negative (SF) and positive (FBS) control data respectively. C. ImageJ-based picture processing. Original pictures were color thresholded to obtain a binary image displaying cellular content as saturated black areas on a white background. Thresholded images were masked to exclude non-cellular particles from the final area calculation. Pictures shown are migrated MDA-MB-231 cells after four hours (<i>top row</i>) and 16 hours (<i>bottom row</i>) of incubation. D. Migratory behavior of MDA-MB-231 cells toward medium+FBS (positive control – <i>filled squares</i>) and background migration (<i>empty squares</i>) as detected by conventional Transwell experiments at ten time points spread over 24 hours of incubation. Area calculation (<i>left</i>) of stained cells and optical density (OD – <i>middle</i>) were compared to the <i>xCELLigence</i> migration pattern, reconstructed from the original high-resolution plot by extrapolating data from the corresponding time points (<i>right</i>). All results represent original data from three independent duplicate experiments ± SD. Picture string (obj. 2.5×) shows migratory status of MDA-MB-231 cells, stained as described, at five different stages during 24 hours of incubation. E. Same as (D) for A549 cells.</p>", "links"=>[], "tags"=>["migratory", "mda-mb-231"], "article_id"=>226163, "categories"=>["Cancer", "Cell Biology", "Developmental Biology", "Biophysics"], "users"=>["Ridha Limame", "An Wouters", "Bea Pauwels", "Erik Fransen", "Marc Peeters", "Filip Lardon", "Olivier De Wever", "Patrick Pauwels"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0046536.g004", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Time_dependent_migratory_pattern_of_MDA_MB_231_and_A549_/226163", "title"=>"Time-dependent migratory pattern of MDA-MB-231 and A549.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-10-19 01:42:43"}

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