Cytometry Part A最新文献

{"title":"High-precision screening and sorting of double emulsion droplets","authors":"Siyuan Zhuang,&nbsp;Lucie Semenec,&nbsp;Stephanie S. Nagy,&nbsp;Amy K. Cain,&nbsp;David W. Inglis","doi":"10.1002/cyto.a.24842","DOIUrl":"10.1002/cyto.a.24842","url":null,"abstract":"<p>Mounting evidence suggests that cell populations are extremely heterogeneous, with individual cells fulfilling different roles within the population. Flow cytometry (FC) is a high-throughput tool for single-cell analysis that works at high optical resolution. Sub-populations with unique properties can be screened, isolated and sorted through fluorescence-activated cell sorting (FACS), using intracellular fluorescent products or surface-tagged fluorescent products of interest. However, traditional FC and FACS methods cannot identify or isolate cells that secrete extracellular products of interest. Double emulsion (DE) droplets are an innovative approach to retaining these extracellular products so cells producing them can be identified and isolated with FC and FACS. The water-in-oil-in-water structure makes DE droplets compatible with the sheath flow of flow cytometry. Single cells can be encapsulated with other reagents into DEs, which act as pico-reactors. These droplets allow biological activities to take place while allowing for cell cultivation monitoring, rare mutant identification, and cellular events characterization. However, using DEs in FACS presents technical challenges, including rupture of DEs, poor accuracy and low sorting efficiency. This study presents high-performance sorting using fluorescent beads (as simulants for cells). This study aims to guide researchers in the use of DE-based flow cytometry, offering insights into how to resolve the technical difficulties associated with DE-based screening and sorting using FC.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 7","pages":"547-554"},"PeriodicalIF":2.5,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24842","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140627244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Setting the gold standard: Commentary on designing and optimizing high-parameter flow cytometry panels","authors":"Stephen C. De Rosa,&nbsp;Yolanda D. Mahnke","doi":"10.1002/cyto.a.24844","DOIUrl":"10.1002/cyto.a.24844","url":null,"abstract":"&lt;p&gt;Technological advances in flow cytometry have greatly expanded its capabilities. These have occurred gradually over time, but there have also been several key advances that have more markedly affected the technology and how it is used. A prime feature of flow cytometry is the ability to characterize cell marker expression at the single-cell level as high-throughput and high volume. The number of cell markers that could be measured simultaneously was initially very low but has been increasing almost exponentially over time. There are many reasons for this increase including advances in hardware for the instrumentation, introduction of new types of fluorescent dyes beyond those found in nature, and also advances in analysis tools that not only enable efficient data analysis but have elegantly allowed for new insights into fluorescent dye/detector interactions that provide theoretical bases for optimal staining panel design in high dimensions.&lt;/p&gt;&lt;p&gt;OMIP-102 [&lt;span&gt;1&lt;/span&gt;] published here represents a milestone in flow cytometry technology and makes use of and expands upon all of those cumulative advances. Simply the demonstration of a “50-color” staining panel is remarkable, but in addition, the approach to the design and the careful and methodical description of the design process both in the main text and the online material provide a definitive syllabus for staining panel design integrating all of the best practices to date.&lt;/p&gt;&lt;p&gt;Because of the wide breadth of information building upon so many of these major advances, it is worthwhile to break down some of those advances into digestible pieces to highlight the significance of this achievement. One key advance in hardware has been the optimization of the instrument optics to enable the relatively weak fluorescent signal to be subdivided most efficiently across large arrays of detectors. Instrument manufacturers have developed their own methods to achieve this goal and the results have been successful with current routine capabilities to detect separate signals from up to 28 fluorescent dyes, and this is expanding. There are multiple Optimized Multicolor Immunofluorescence Panel (OMIP) publications demonstrating successful staining panels at this scale. Likely the most significant advance representing a new paradigm is spectral optics. This is a brilliant concept that in retrospect seems so obvious as the likely best approach. While full spectrum cytometry was first demonstrated in 2004 [&lt;span&gt;2, 3&lt;/span&gt;], it required further hardware and software advances to enable routine implementation by a wide user base.&lt;/p&gt;&lt;p&gt;The potential of exploiting the light spectrum more completely for interrogating fluorescently labeled biological specimens directly called for the development of new fluorescent dyes in order to make high-parameter flow cytometry a reality. As the discovery of natural fluorochromes with the appropriate brightness and spectral characteristics was limiting, luckily, custom-designed","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 6","pages":"428-429"},"PeriodicalIF":3.7,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24844","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140627191","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Volume 105A, Number 4, April 2024 Cover Image","authors":"","doi":"10.1002/cyto.a.24748","DOIUrl":"https://doi.org/10.1002/cyto.a.24748","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 4","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24748","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140606360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A flow cytometry-based assay to measure neutralizing antibodies against SARS-CoV-2 virus","authors":"Veridiane M. Pscheidt,&nbsp;Priscila Oliveira de Souza,&nbsp;Tiago Fazolo,&nbsp;José Luiz Proença Modena,&nbsp;Camila Simeoni,&nbsp;Daniel Teixeira,&nbsp;Natália Brunetti Silva,&nbsp;Karina Bispo dos Santos,&nbsp;Luiz Rodrigues Júnior,&nbsp;Cristina Bonorino","doi":"10.1002/cyto.a.24838","DOIUrl":"10.1002/cyto.a.24838","url":null,"abstract":"<p>The COVID-19 pandemic caused by the SARS-CoV-2 virus has highlighted the need for serological assays that can accurately evaluate the neutralizing efficiency of antibodies produced during infection or induced by vaccines. However, conventional assays often require the manipulation of live viruses on a level-three biosafety (BSL3) facility, which presents practical and safety challenges. Here, we present a novel, alternative assay that measures neutralizing antibodies (NAbs) against SARS-CoV-2 in plasma using flow cytometry. This assay is based on antibody binding to the S protein and has demonstrated precision in both intra- and inter-assay measurements at a dilution of 1:50. The cut-off was determined using Receiver Operating Characteristic (ROC) analysis and the value of 36.01% has shown high sensitivity and specificity in distinguishing between pre-pandemic sera, COVID-19 patients, and vaccinated individuals. The efficiency significantly correlates with the gold standard test, PRNT. Our new assay offers a safe and efficient alternative to conventional assays for evaluating NAbs against SARS-CoV-2.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 6","pages":"446-457"},"PeriodicalIF":3.7,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140595431","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive data analysis of white blood cells with classification and segmentation by using deep learning approaches","authors":"Şeyma Nur Özcan,&nbsp;Tansel Uyar,&nbsp;Gökay Karayeğen","doi":"10.1002/cyto.a.24839","DOIUrl":"10.1002/cyto.a.24839","url":null,"abstract":"<p>Deep learning approaches have frequently been used in the classification and segmentation of human peripheral blood cells. The common feature of previous studies was that they used more than one dataset, but used them separately. No study has been found that combines more than two datasets to use together. In classification, five types of white blood cells were identified by using a mixture of four different datasets. In segmentation, four types of white blood cells were determined, and three different neural networks, including CNN (Convolutional Neural Network), UNet and SegNet, were applied. The classification results of the presented study were compared with those of related studies. The balanced accuracy was 98.03%, and the test accuracy of the train-independent dataset was determined to be 97.27%. For segmentation, accuracy rates of 98.9% for train-dependent dataset and 92.82% for train-independent dataset for the proposed CNN were obtained in both nucleus and cytoplasm detection. In the presented study, the proposed method showed that it could detect white blood cells from a train-independent dataset with high accuracy. Additionally, it is promising as a diagnostic tool that can be used in the clinical field, with successful results in classification and segmentation.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 7","pages":"501-520"},"PeriodicalIF":2.5,"publicationDate":"2024-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24839","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140335113","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Progress and challenges of in vivo flow cytometry and its applications in circulating cells of eyes","authors":"Wei Lin,&nbsp;Peng Wang,&nbsp;Yingxin Qi,&nbsp;Yanlong Zhao,&nbsp;Xunbin Wei","doi":"10.1002/cyto.a.24837","DOIUrl":"10.1002/cyto.a.24837","url":null,"abstract":"<p>Circulating inflammatory cells in eyes have emerged as early indicators of numerous major diseases, yet the monitoring of these cells remains an underdeveloped field. In vivo flow cytometry (IVFC), a noninvasive technique, offers the promise of real-time, dynamic quantification of circulating cells. However, IVFC has not seen extensive applications in the detection of circulating cells in eyes, possibly due to the eye's unique physiological structure and fundus imaging limitations. This study reviews the current research progress in retinal flow cytometry and other fundus examination techniques, such as adaptive optics, ultra-widefield retinal imaging, multispectral imaging, and optical coherence tomography, to propose novel ideas for circulating cell monitoring.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 6","pages":"437-445"},"PeriodicalIF":3.7,"publicationDate":"2024-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140317979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorochrome-dependent specific changes in spectral profiles using different compensation beads or primary cells in full spectrum cytometry","authors":"Yaroslava Shevchenko,&nbsp;Isabella Lurje,&nbsp;Frank Tacke,&nbsp;Linda Hammerich","doi":"10.1002/cyto.a.24836","DOIUrl":"10.1002/cyto.a.24836","url":null,"abstract":"<p>Full spectrum flow cytometry is a powerful tool for immune monitoring on a single-cell level and with currently available machines, panels of 40 or more markers per sample are possible. However, with an increased panel size, spectral unmixing issues arise, and appropriate single stain reference controls are required for accurate experimental results and to avoid unmixing errors. In contrast to conventional flow cytometry, full spectrum flow cytometry takes into account even minor differences in spectral signatures and requires the full spectrum of each fluorochrome to be identical in the reference control and the fully stained sample to ensure accurate and reliable results. In general, using the cells of interest is considered optimal, but certain markers may not be expressed at sufficient levels to generate a reliable positive control. In this case, compensation beads show some significant advantages as they bind a consistent amount of antibody independent of its specificity. In this study, we evaluated two types of manufactured compensation beads for use as reference controls for 30 of the most commonly used and commercially available fluorochromes in full spectrum cytometry and compared them to human and murine primary leukocytes. While most fluorochromes show the same spectral profile on beads and cells, we demonstrate that specific fluorochromes show a significantly different spectral profile depending on which type of compensation beads is used, and some fluorochromes should be used on cells exclusively. Here, we provide a list of important considerations when selecting optimal reference controls for full spectrum flow cytometry.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 6","pages":"458-463"},"PeriodicalIF":3.7,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24836","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140174104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Volume 105A, Number 3, March 2024 Cover Image","authors":"","doi":"10.1002/cyto.a.24746","DOIUrl":"https://doi.org/10.1002/cyto.a.24746","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24746","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140123735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Automated antibody dispensing to improve high-parameter flow cytometry throughput and analysis","authors":"Victor Bosteels,&nbsp;Julie Van Duyse,&nbsp;Elien Ruyssinck,&nbsp;Katrien Van der Borght,&nbsp;Long Nguyen,&nbsp;Jannes Gavel,&nbsp;Sophie Janssens,&nbsp;Gert Van Isterdael","doi":"10.1002/cyto.a.24835","DOIUrl":"10.1002/cyto.a.24835","url":null,"abstract":"<p>Over the past decade, the flow cytometry field has witnessed significant advancements in the number of fluorochromes that can be detected. This enables researchers to analyze more than 40 markers simultaneously on thousands of cells per second. However, with this increased complexity and multiplicity of markers, the manual dispensing of antibodies for flow cytometry experiments has become laborious, time-consuming, and prone to errors. An automated antibody dispensing system could provide a potential solution by enhancing the efficiency, and by improving data quality by faithfully dispensing the fluorochrome-conjugated antibodies and by enabling the easy addition of extra controls. In this study, a comprehensive comparison of different liquid handlers for dispensing fluorochrome-labeled antibodies was conducted for the preparation of flow cytometry stainings. The evaluation focused on key criteria including dispensing time, dead volume, and reliability of dispensing. After benchmarking, the I.DOT, a non-contact liquid handler, was selected and optimized in more detail. In the end, the I.DOT was able to prepare a 25-marker panel in 20 min, including the full stain, all FMOs and all single stain controls for cells and beads. Having all these controls improved the validation of the panel, visualization, and analysis of the data. Thus, automated antibody dispensing by dispensers such as the I.DOT reduces time and errors, enhances data quality, and can be easily integrated in an automated workflow to prepare samples for flow cytometry.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 6","pages":"464-473"},"PeriodicalIF":3.7,"publicationDate":"2024-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24835","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140058907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unveiling the epigenetic landscape of plants using flow cytometry approach","authors":"Thakur Prava Jyoti,&nbsp;Shivani Chandel,&nbsp;Rajveer Singh","doi":"10.1002/cyto.a.24834","DOIUrl":"10.1002/cyto.a.24834","url":null,"abstract":"<p>Plants are sessile creatures that have to adapt constantly changing environmental circumstances. Plants are subjected to a range of abiotic stressors as a result of unpredictable climate change. Understanding how stress-responsive genes are regulated can help us better understand how plants can adapt to changing environmental conditions. Epigenetic markers that dynamically change in response to stimuli, such as DNA methylation and histone modifications are known to regulate gene expression. Individual cells or particles' physical and/or chemical properties can be measured using the method known as flow cytometry. It may therefore be used to evaluate changes in DNA methylation, histone modifications, and other epigenetic markers, making it a potent tool for researching epigenetics in plants. We explore the use of flow cytometry as a technique for examining epigenetic traits in this thorough discussion. The separation of cell nuclei and their subsequent labeling with fluorescent antibodies, offering information on the epigenetic mechanisms in plants when utilizing flow cytometry. We also go through the use of high-throughput data analysis methods to unravel the complex epigenetic processes occurring inside plant systems.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 4","pages":"231-241"},"PeriodicalIF":3.7,"publicationDate":"2024-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140027582","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}