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• W2885550008 abstract "•TNNI3 and TNNT2 proteins are present in day 25 hPSC-CMs•Commonly used reagents can lead to non-specific binding of anti-troponin antibodies•A fit-for-purpose workflow describes how to develop a flow cytometry protocol•A robust protocol for routine quality control testing was validated for hPSC-CMs Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation. Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation. Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes (hPSC-CMs) offers an inexhaustible supply of cells for basic science research and translational applications, including drug testing (Braam et al., 2010Braam S.R. Tertoolen L. van de Stolpe A. Meyer T. Passier R. Mummery C.L. et al.Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes.Stem Cell Res. 2010; 4: 107-116Crossref PubMed Scopus (313) Google Scholar), disease modeling (Carvajal-Vergara et al., 2010Carvajal-Vergara X. Sevilla A. D'Souza S.L. Ang Y.S. Schaniel C. Lee D.F. Yang L. Kaplan A.D. Adler E.D. Rozov R. et al.Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.Nature. 2010; 465: 808-812Crossref PubMed Scopus (573) Google Scholar, Moretti et al., 2010Moretti A. Bellin M. Welling A. Jung C.B. Lam J.T. Bott-Flügel L. Dorn T. Goedel A. Höhnke C. Hofmann F. et al.Patient-specific induced pluripotent stem-cell models for long-QT syndrome.N. Engl. J. Med. 2010; 363: 1397-1409Crossref PubMed Scopus (961) Google Scholar), and regenerative medicine (Chong et al., 2014Chong J.J. Yang X. Don C.W. Minami E. Liu Y.W. Weyers J.J. Mahoney W.M. Van Biber B. Cook S.M. Palpant N.J. et al.Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.Nature. 2014; 510: 273-277Crossref PubMed Scopus (943) Google Scholar, van Laake et al., 2007van Laake L.W. Passier R. Monshouwer-Kloots J. Verkleij A.J. Lips D.J. Freund C. den Ouden K. Ward-van Oostwaard D. Korving J. Tertoolen L.G. van Echteld C.J. et al.Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction.Stem Cell Res. 2007; 1: 9-24Crossref PubMed Scopus (317) Google Scholar). Using modern differentiation protocols, hPSC-CMs can be efficiently generated from human embryonic stem (hESC) and human induced pluripotent stem (hiPSC) cells, which has led to an increasing number of laboratories and studies implementing this technology (reviewed in Batalov and Feinberg, 2015Batalov I. Feinberg A.W. Differentiation of cardiomyocytes from human pluripotent stem cells using monolayer culture.Biomark. Insights. 2015; 10: 71-76PubMed Google Scholar, Mummery et al., 2012Mummery C.L. Zhang J. Ng E.S. Elliott D.A. Elefanty A.G. Kamp T.J. et al.Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview.Circ. Res. 2012; 111: 344-358Crossref PubMed Scopus (509) Google Scholar). However, despite significant advancements in defining the factors most critical for cardiomyogenic differentiation (Burridge et al., 2014Burridge P.W. Matsa E. Shukla P. Lin Z.C. Churko J.M. Ebert A.D. Lan F. Diecke S. Huber B. Mordwinkin N.M. et al.Chemically defined generation of human cardiomyocytes.Nat. Methods. 2014; 11: 855-860Crossref PubMed Google Scholar, Lian et al., 2015Lian X. Bao X. Zilberter M. Westman M. Fisahn A. Hsiao C. Hazeltine L.B. Dunn K.K. Kamp T.J. Palecek S.P. et al.Chemically defined, albumin-free human cardiomyocyte generation.Nat. Methods. 2015; 12: 595-596Crossref PubMed Scopus (105) Google Scholar), the resulting cultures remain a heterogeneous mixture with regard to cell type and subtype, and this heterogeneity can be exacerbated by variations among cell lines, protocols, and personnel (Ohno et al., 2013Ohno Y. Yuasa S. Egashira T. Seki T. Hashimoto H. Tohyama S. Saito Y. Kunitomi A. Shimoji K. Onizuka T. et al.Distinct iPS cells show different cardiac differentiation efficiency.Stem Cells Int. 2013; 2013: 659739Crossref PubMed Scopus (11) Google Scholar). Ultimately, as heterogeneity can pose challenges to interpreting functional data, the ability to accurately and precisely assess cell identity in differentiation cultures is paramount to well-defined and reproducible studies. Flow cytometry is a quantitative, cell population-based single-cell approach to assess individual cell phenotypes, rendering it an ideal strategy for assessment of hPSC-CM heterogeneity. In this approach, population heterogeneity is typically assessed based on detection of endogenous proteins by specific monoclonal antibodies or expression of exogenous marker proteins driven by cell- or tissue-restricted promoters. Considering the availability of benchtop cytometers and prevalence of flow cytometry core facilities at most research organizations, this approach is affordable and accessible to most laboratories. Altogether, flow cytometry is well suited to use in routine quality control assessments of hPSC-CM cultures. The proper implementation of flow cytometry requires optimization of many procedural parameters within sample preparation, data acquisition, and data analysis. Examples include optimizing the cell collection method to produce single-cell suspensions, validating monoclonal antibody specificity, titrating antibody concentrations, selecting appropriate negative and positive controls, adjusting cytometer laser settings, and developing acceptable gating strategies. Considering the numerous procedural variables, this optimization process can be daunting. Unfortunately, a standardized and validated protocol that is broadly applicable among laboratories has not been established for assessing cardiomyocyte identity within hPSC-CM cultures. Consequently, accurate comparisons of outcomes generated by various differentiation protocols or cell lines among laboratories and studies, including assessments of purity, reproducibility, and functional data, remain challenging. A survey of studies published over the past 7 years (1/2010–10/2017) reveals a wide range of antibodies and experimental conditions reported for flow cytometry-based assessment of hPSC-CMs. Of the 84 studies that use flow cytometry, the majority (n = 68) used cardiac troponin T (TNNT2) as the primary marker to assess hPSC-CM cultures (Figure 1A). Of these studies, nearly 72% used one of two monoclonal antibodies (clone 13-11 or 1C11), and 28% used a variety of other antibodies, including monoclonal and polyclonal, to detect TNNT2. Of concern, 18% of TNNT2 studies failed to report either the antibody clone or the vendor or both. The sample preparation conditions among studies were more disparate (Figure 1B), with nine fixation and fifteen permeabilization conditions reported. Moreover, many studies failed to report the relevant details for fixation (>15%) and permeabilization (>26%). Altogether, there is currently no consensus regarding which marker, antibody, or protocol is best suited to enable comparisons of hPSC-CM culture heterogeneity among experiments or laboratories. Considering the lack of consensus regarding marker, antibody, and protocol, the broad goals of this study were to evaluate antibody specificity and sample preparation conditions for the assessment of cardiomyocyte identity within hPSC-CM cultures by flow cytometry. Three sample preparation methods in conjunction with five commercially available anti-cardiac troponin I (TNNI3) and two anti-TNNT2 antibodies were applied to hPSC-CMs and two negative control cell types, undifferentiated hPSCs and cardiac fibroblasts. In performing these analyses, we found that the choice of fixation protocol and antibody had significant and variable effects on the accuracy of cardiomyocyte identity assessment. It is expected that by providing details regarding validation of antibody specificity within this context and revealing pitfalls with commonly used antibodies and preparation conditions, these results will benefit laboratories with established expertise in hPSC-CM differentiation as well as those new to this field. By establishing rigorous standards for quality control evaluation of hPSC-CMs, we believe that the approaches described here will facilitate the use of hPSC-CMs in a broad range of research and clinical applications, especially by enabling more accurate comparisons of results among studies. To facilitate data sharing among laboratories, the current study aims to set a standard regarding the experimental details that should be included when publishing flow cytometry-based assessments of hPSC-CMs, consistent with similar calls for publication guidelines (Lee et al., 2008Lee J.A. Spidlen J. Boyce K. Cai J. Crosbie N. Dalphin M. Furlong J. Gasparetto M. Goldberg M. Goralczyk E.M. et al.MIFlowCyt: the minimum information about a flow cytometry experiment.Cytometry A. 2008; 73: 926-930Crossref PubMed Scopus (327) Google Scholar). Finally, based on the results of the current study, a comprehensive protocol for assessment of cardiomyocyte identity in hPSC-CM cultures by flow cytometry is provided. The protocol provides stepwise instructions and describes key points to consider for sample preparation and antibody validation, with the expectation that providing these details will facilitate its use among laboratories. As this protocol has been successfully replicated in three different laboratories and can be completed in less than 3 h, from adherent-cell collection to data analysis, it is suitable for routine assessment of hPSC-CM cultures. The expression of troponin complex components is temporally regulated during normal human development in a tissue-specific manner (Bhavsar et al., 1991Bhavsar P.K. Dhoot G.K. Cumming D.V. Butler-Browne G.S. Yacoub M.H. Barton P.J. et al.Developmental expression of troponin I isoforms in fetal human heart.FEBS Lett. 1991; 292: 5-8Crossref PubMed Scopus (88) Google Scholar, Hunkeler et al., 1991Hunkeler N.M. Kullman J. Murphy A.M. et al.Troponin I isoform expression in human heart.Circ. Res. 1991; 69: 1409-1414Crossref PubMed Scopus (152) Google Scholar, Sasse et al., 1993Sasse S. Brand N.J. Kyprianou P. Dhoot G.K. Wade R. Arai M. Periasamy M. Yacoub M.H. Barton P.J. et al.Troponin I gene expression during human cardiac development and in end-stage heart failure.Circ. Res. 1993; 72: 932-938Crossref PubMed Scopus (204) Google Scholar). While similar trends in temporal regulation have been reported for in vitro differentiation of hPSC-CMs, discrepancy with regard to the timing of the emergence of TNNI3 has been reported, with one report suggesting it emerges after months in culture (Bedada et al., 2014Bedada F.B. Chan S.S. Metzger S.K. Zhang L. Zhang J. Garry D.J. Kamp T.J. Kyba M. Metzger J.M. et al.Acquisition of a quantitative, stoichiometrically conserved ratiometric marker of maturation status in stem cell-derived cardiac myocytes.Stem Cell Reports. 2014; 3: 594-605Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and others showing it is expressed as early as day 8 (Puppala et al., 2013Puppala D. Collis L.P. Sun S.Z. Bonato V. Chen X. Anson B. Pletcher M. Fermini B. Engle S.J. et al.Comparative gene expression profiling in human-induced pluripotent stem cell–derived cardiocytes and human and cynomolgus heart tissue.Toxicol. Sci. 2013; 131: 292-301Crossref PubMed Scopus (36) Google Scholar, Tompkins et al., 2016Tompkins J.D. Jung M. Chen C.Y. Lin Z. Ye J. Godatha S. Lizhar E. Wu X. Hsu D. Couture L.A. Riggs A.D. et al.Mapping human pluripotent-to-cardiomyocyte differentiation: methylomes, transcriptomes, and exon DNA methylation “memories”.EBioMedicine. 2016; 4: 74-85Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). For this reason, we used a targeted mass spectrometry approach to confirm the presence of TNNI3 and TNNT2 protein in day 25 hPSC-CMs as a first step in the selection of reliable markers of cardiomyocyte identity. The approach, parallel reaction monitoring (PRM), uses high-resolution/accurate mass instrumentation to specifically detect pre-selected peptides within a mixture (Peterson et al., 2012Peterson A.C. Russell J.D. Bailey D.J. Westphall M.S. Coon J.J. et al.Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics.Mol. Cell. Proteomics. 2012; 11: 1475-1488Crossref PubMed Scopus (819) Google Scholar). Here, PRM assays were developed to specifically detect three unique peptides from TNNI3 and three from TNNT2. Stable isotopically labeled peptides for TNNI3 were included as internal controls to provide added rigor for this protein because of reported discrepancies regarding the timing of its expression. Application of this PRM assay reliably detected peptides from both TNNI3 and TNNT2 in day 25 hPSC-CMs and in human cardiac tissue, but not in undifferentiated hPSCs (Figures 2 and S1). Importantly, for TNNI3 peptides, the endogenous and isotopically labeled peptides co-eluted and had identical fragmentation patterns across hPSC-CMs and cardiac tissue, providing unequivocal evidence that this protein is present in these samples (Figure 2). Altogether, this highly sensitive, antibody-independent mass spectrometry strategy confirms the presence of TNNI3 and TNNT2 in day 25 hPSC-CMs produced by the differentiation protocol used here. Although our literature survey revealed that a preponderance of studies relied on TNNT2 as a marker of cardiomyocyte identity, TNNI3 is more specific to cardiomyocytes than TNNT2 throughout human development. As the mass spectrometry analysis confirmed the presence of both proteins in day 25 hPSC-CMs, antibodies to both TNNI3 and TNNT2 were investigated for their ability to serve as markers of cardiomyocyte identity within hPSC-CM cultures. The two most common troponin T2 antibodies from previous studies (Figure 1A) and five anti-TNNI3 antibodies whose epitopes span the range of the amino acid sequence for TNNI3 (Figure 1C) were assessed for their ability to specifically detect hPSC-CMs using three different sample preparation conditions (Table 1). In the initial screen, all seven antibodies were assessed for their ability to produce signal stronger than that of an equivalent amount of isotype control and to distinguish hPSC-CMs from undifferentiated hPSCs, a relevant negative cell-type control. All data for two biological replicate analyses of each clone and the sample preparation protocol are presented in Figures S2A–S2G. Overall, flow cytometry results were highly dependent on sample preparation conditions for some antibodies, and less so for other clones (summarized in Figure 3A, supporting data in Figure S3). For example, all three sample preparation protocols yielded satisfactory results for clone 1C11, but the ability to distinguish between positive and negative cell types was protocol dependent for clones 13-11 and 2Q1100 (Figure 3B). Clone 19C7 failed to produce desirable results independent of protocol, as it produced a stronger signal in the negative cell type control than in hPSC-CMs (Figure 3B). Each sample preparation strategy can produce samples suitable for flow cytometry as demonstrated by single-cell suspensions that were separable from debris by gating on forward (FSC) and side scatter (SSC) (Figure S2), and scatterplots for all subsequent experiments were comparable with those shown in Figure S2. However, samples prepared using protocol 2 exhibited more favorable handling characteristics (i.e., a tight, visible cell pellet) and, in general, better resolution compared with protocols 1 and 3. Two doublet-exclusion gating strategies were compared with the single SSC area versus FSC area gating strategy and were not found to substantially alter the observed percentage positivity or mean intensity of negative and positive populations (Figure S3). Consequently, protocol 2 and the four antibodies (1C11, 13–11, C5, 2Q1100) that provided the most satisfactory results during the initial screen were assessed further in subsequent experiments using the single gating strategy.Table 1Summary of the Experimental Conditions Examined for Their Suitability for Assessing hPSC-CM Cultures by Flow CytometryProtocol DetailsReagentsProtocol 1Protocol 2Protocol 3FixationBD Cytofix2% formaldehyde (w/v) in DPBS−/−2% formaldehyde (w/v) in DPBS−/−PermeabilizationBD Perm Buffer III0.5% saponin (w/v) in block0.1% Triton X-100 (w/v) in blockBlocking/antibody bindingblock solutionblock solutionblock solutionResuspensionProtocol details1. Fixation15 min, on ice20 min20 min2. Wash2 × 3 mL2 × 3 mL2 × 3 mL3. Permeabilization30 min, on icePerformed as one 15 min incubation15 min4. Wash2 × 3 mL2 × 3 mL5. Block15 min, on ice15 min6. 1° antibody45 min, on ice45 min45 min7. Wash2 × 3 mL2 × 3 mL2 × 3 mL8. 2° antibody (if applicable)30 min, on ice30 min30 min9. Wash (if applicable)2 × 3 mL2 × 3 mL2 × 3 mL10. Resuspension500 μL500 μL500 μLInstrument configurationsInstrumentBD LSR IILaser lines488 nm (50 mW)562 nm (100 mW)640 nm (100 mW)Emission filters525/50585/15670/30FluorochromeFITC/Alexa Fluor 488PEAPCAntibody DetailsClone1° Ab vendorCatalog no.FluorophoreAmount of 1° Ab (μg)2° Ab2° Ab vendorCatalog no.Amount of 2° Ab (μg)TNNT213-11Thermo FisherMA512960—0.1anti-mouseIgG1-AF 488Thermo Fisher A211210.61C11Abcam ab8295FITC1.0———TNNI3EP1106YOrigeneTA303719—1.0anti-rabbitIgG-AF 488Thermo Fisher A110080.619C7Abcam ab19615—1.0anti-mouse IgG2b-AF 488Thermo Fisher A211410.64C2Fitzgerald10R-T123e—1.0anti-mouse IgG2a-AF 488Thermo Fisher A211310.6C5Fitzgerald10R-T123k—1.0anti-mouse IgG2b-AF 488/AF 647Thermo Fisher A21141/A212420.62Q1100US BiologicalT8665-13FPE0.5———Details are provided for the three sample preparation protocols, the seven antibodies evaluated, and the flow cytometer instrument configurations. Block solution, 0.5% w/v BSA in DPBS−/−; wash solution, DPBS−/−; BD, Becton Dickinson; Ab, antibody; AF, Alexa Fluor; FITC, fluorescein isothiocyanate. Open table in a new tab Details are provided for the three sample preparation protocols, the seven antibodies evaluated, and the flow cytometer instrument configurations. Block solution, 0.5% w/v BSA in DPBS−/−; wash solution, DPBS−/−; BD, Becton Dickinson; Ab, antibody; AF, Alexa Fluor; FITC, fluorescein isothiocyanate. Four concentrations were tested for each antibody, based either on vendor recommendations or on the results of the screen, to determine the optimal concentration for providing a maximal separation in signal between positive and negative cell types (Figures 4A and S4). The performance of all four antibodies was consistent with results expected for a successful titration study (i.e., signal dependent on antibody concentration that eventually becomes saturated in a positive population) (Figures 4A and S4). Three clones (1C11, 2Q1100, and C5) that were best able to distinguish between negative and positive populations were selected for further validation using the optimal antibody amount determined by titration: 0.5 μg for 1C11 and 2Q1100, 0.1 μg for C5. The specificity of clones 1C11, 2Q1100, and C5 for their reported epitopes was assessed using a competition assay in which signal from each naive antibody was compared with antibody pre-incubated with peptide antigen. In this manner, a diminution or ablation of signal caused by incubation with peptide antigen can be indicative of specificity for the reported epitope. Due to the high sequence identity between the isoforms of TNNI1, TNNI2, and TNNI3 at the reported epitope for both clones C5 and 2Q1100 (Figure 1C), the homologous peptide regions to the TNNI3 epitope were also investigated. Antibodies incubated with TNNT2 and TNNI3 epitopes were included as negative controls for anti-TNNI3 and anti-TNNT2, respectively, for these experiments. Amino acid sequences for purified peptide antigens are shown in Figure 1C. TNNI1, TNNI2, and TNNI3 peptides were able to partially block anti-TNNI3 clone 2Q1100 binding to hPSC-CMs as shown by the overall decrease in fluorescence intensity and collapse of the histogram into a unimodal distribution. In contrast, these peptides had only a minor effect on binding of anti-TNNI3 clone C5 to hPSC-CMs (Figures 4B and S5A). Anti-TNNT2 clone 1C11 antigen peptide similarly had no effect on the fluorescent signal compared with naive antibody (Figure S5A). Although the epitope competition assay was unable to unequivocally verify specificity of the antibodies for their reported peptide epitopes, this may be simply due to a linear peptide lacking the necessary secondary or tertiary structure of the native epitope. Consequently, a co-immunodetection strategy was used to determine if antibodies to TNNI3 and TNNT2 were specific to the same cell population as an alternative assessment of specificity. If they do not overlap this would suggest that one or both are binding to non-cardiomyocytes. When hPSC-CMs were evaluated with anti-TNNI3 and anti-TNNT2 antibodies using the clone pairs 2Q1100/1C11 and C5/1C11, less than 3% of the population, on average, was positive for only a single antibody (i.e., 97% of cells were positive for both antibodies or for neither). These results demonstrate that, under these preparation conditions, the TNNI3 and TNNT2 antibodies used here mark the same cell population (Figures 4C and S5B). These results, together with the observation that immunofluorescence imaging experiments using anti-TNNT2 clone 1C11 yield a striated localization pattern expected for a sarcomere protein (Figure S5C), support that these antibodies are specifically detecting their respective protein targets when used with this sample preparation protocol. In addition, while the application of this protocol within the context of hPSC differentiation was the focus of this study, the protocol was applied to cardiac fibroblasts, a biologically relevant negative cell type, in co-culture (Thavandiran et al., 2013Thavandiran N. Dubois N. Mikryukov A. Massé S. Beca B. Simmons C.A. Deshpande V.S. McGarry J.P. Chen C.S. Nanthakumar K. et al.Design and formulation of functional pluripotent stem cell-derived cardiac microtissues.Proc. Natl. Acad. Sci. U S A. 2013; 110: E4698-E4707Crossref PubMed Scopus (202) Google Scholar) and trans-differentiation experiments (Addis et al., 2013Addis R.C. Ifkovits J.L. Pinto F. Kellam L.D. Esteso P. Rentschler S. Christoforou N. Epstein J.A. Gearhart J.D. et al.Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.J. Mol. Cell. Cardiol. 2013; 60: 97-106Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, Fu et al., 2013Fu J.D. Stone N.R. Liu L. Spencer C.I. Qian L. Hayashi Y. Delgado-Olguin P. Ding S. Bruneau B.G. Srivastava D. et al.Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.Stem Cell Reports. 2013; 1: 235-247Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Overall, using this protocol, all three antibody clones generated histograms from cardiac fibroblasts that were indistinguishable from isotype control (Figures 4D and S5D). Finally, this protocol was found to be applicable to earlier (day 10) and later (day 95) time points of differentiation cultures (Figure S5E). To accurately determine the percentage of cardiomyocytes within a heterogeneous hPSC-CM culture, a protocol, including antibody and all experimental conditions, must be able to distinguish cardiomyocytes from non-cardiomyocytes within a single tube. To evaluate the best-performing protocol for this capacity, three antibody clones (1C11, 2Q1100, and C5) were used in conjunction with protocol 2 to assess population heterogeneity within samples where hPSC-CMs and hPSCs were mixed at various ratios (100:0, 75:25, 50:50, 25:75, and 0:100). Overall, each antibody clone in conjunction with sample preparation protocol 2 can distinguish between positive and negative cell types (Figures 5 and S6). At each ratio of hPSC-CM to hPSC, a bimodal population was observed where the percentage of positive cells decreased in proportion to the number of hPSCs added to the sample (Figures 5 and S6). The percent positivity observed for samples that were a mix of hPSC-CMs and hPSCs correlated well with the expected percentages calculated based on the unmixed sample, averaging less than an 8% difference. Deviations from expected percentages are likely due to variations in cell counting as evidenced by dissimilarities in the event rates observed on the flow cytometer (∼700 events/s for 100% hPSC versus ∼475 events/s for 100% hPSC-CM differentiation culture) and by the increase in the percentage errors that correlated with the amount of hPSCs added (Figures 5 and S6). Considering the strong performance of these antibodies and protocol in cell mixing experiments, a detailed standard operating procedure was established and shared with two laboratories located in different institutions to further test rigor and reproducibility. Results from these two laboratories were comparable with our own data, despite using different cell lines and differentiation protocols, and similar trends were observed for correlations between expected and measured percentage positivity and the maintenance of a bimodal population across samples (Figures 5 and S6). The spirit of this study is responsive to calls for improving scientific rigor and reproducibility discussed in several recent publications (Bordeaux et al., 2010Bordeaux J. Welsh A. Agarwal S. Killiam E. Baquero M. Hanna J. Anagnostou V. Rimm D. et al.Antibody validation.Biotechniques. 2010; 48: 197-209Crossref PubMed Scopus (447) Google Scholar, Bradbury and Pluckthun, 2015Bradbury A. Pluckthun A. Reproducibility: standardize antibodies used in research.Nature. 2015; 518: 27-29Crossref PubMed Scopus (440) Google Scholar, Brooks and Lindsey, 2018Brooks H.L. Lindsey M.L. Guidelines for authors and reviewers on antibody use in physiology studies.Am. J. Physiol. Heart Circ. Physiol. 2018; 314: H724-H732Crossref PubMed Scopus (49) Google Scholar) and reflected in policies for reagent validation that are now required by granting agencies (e.g., NIH). Our success in developing a replicable protocol supported the development of a standardized workflow for rigorous selection and evaluation of antibodies and sample preparation conditions for flow cytometry experiments (Figure 6). This workflow outlines major steps required to establish the fit-for-purpose of an antibody and protocol for assessing cell population identity within a heterogeneous mixture. To be clear, although data from vendors or previous publications can serve as starting points, antibody validation is ultimately the responsibility of the user and should be performed for each antibody clone, cell type, and protocol. To begin, suitable markers can be selected from the literature or experimentally determined by using mass spectrometry. The superior selectivity, sensitivity, and specificity of targeted mass spectrometry make it an ideal technique for verifying the presence of candidate markers in cell types of interest compared with antibody-based techniques such as immunoblotting (Aebersold et al., 2013Aebersold R. Burlingame A.L. Bradshaw R.A. et al.Western blots versus selected reaction monitoring assays: time to turn the tables?.Mol. Cell. Proteomics. 2013; 12: 2381-2382Crossref PubMed Scopus (204) Google Scholar). The selection of antibody clones should consider published literature and vendor data as well as specific information about the epitope, including uniqueness of the sequence and possible variants or post-translational modifications. As exemplified in this study, it is advisable to test more than one antibody clone and more tha" @default.
• W2885550008 created "2018-08-22" @default.
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• W2885550008 date "2019-02-01" @default.
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• W2885550008 title "Are These Cardiomyocytes? Protocol Development Reveals Impact of Sample Preparation on the Accuracy of Identifying Cardiomyocytes by Flow Cytometry" @default.
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• W2885550008 doi "https://doi.org/10.1016/j.stemcr.2018.12.016" @default.
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