Cytometry Part A最新文献

{"title":"Volume 105A, Number 12, December 2024 Cover Image","authors":"","doi":"10.1002/cyto.a.24764","DOIUrl":"https://doi.org/10.1002/cyto.a.24764","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 12","pages":""},"PeriodicalIF":2.5,"publicationDate":"2024-12-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24764","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142861233","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":"Autofluorescence lifetime flow cytometry rapidly flows from strength to strength.","authors":"Klaus Suhling","doi":"10.1002/cyto.a.24909","DOIUrl":"https://doi.org/10.1002/cyto.a.24909","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":""},"PeriodicalIF":2.5,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142767366","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":"Flow cytometry-based method to detect and separate Mycoplasma hyorhinis in cell cultures.","authors":"Chunzhuo Liu, Hui Wang, Shan Liu, Mengyuan Li","doi":"10.1002/cyto.a.24908","DOIUrl":"https://doi.org/10.1002/cyto.a.24908","url":null,"abstract":"<p><p>Mycoplasma hyorhinis is a frequently observed contaminant in cell cultures, and its detection and purification pose considerable challenges. Fragments or other cell components are similar in size to those of Mycoplasma; therefore, distinguishing them is difficult. In this study, we used Hoechst staining in combination with carboxyfluorescein succinimidyl ester (CFSE) to label Mycoplasma. The trigger threshold was set in the Hoechst Blue channel rather than in the default forward scatter channel, utilizing the differences in DNA content between Mycoplasma and fragments. Subsequently, we identified and isolated it at single-cell resolution via flow cytometry and successfully sorted infectious Mycoplasma in cell culture. Simultaneously, we validated the accuracy and feasibility of this approach using polymerase chain reaction, fluorescence confocal microscopy, and cryo-electron microscopy. This methodology enabled the diagnosis of Mycoplasma at extremely low concentrations, significantly enhancing the detection efficiency and facilitating the isolation and purification of parasitic Mycoplasma in cell culture instead of pure Mycoplasma culture in artificial media for subsequent studies.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":" ","pages":""},"PeriodicalIF":2.5,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142767370","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":"The consequence of mismatched buffers in purity checks when spectral cell sorting","authors":"Rita A. S. Dapaah,&nbsp;Laura Ferrer-Font,&nbsp;Xiaoshan Shi,&nbsp;Christopher Hall,&nbsp;Sam Thompson,&nbsp;Larissa Catharina Costa,&nbsp;Peter L. Mage,&nbsp;Aaron J. Tyznik,&nbsp;Kelly Lundsten,&nbsp;Rachael V. Walker","doi":"10.1002/cyto.a.24911","DOIUrl":"10.1002/cyto.a.24911","url":null,"abstract":"<p>Although spectral flow cytometry has become a ubiquitous tool for cell analysis, the use of spectral cytometry on cell sorters requires additional considerations arising from the unique requirements of sorting workflows. Here, we show that care should be taken when ascertaining the purity of a sort on a spectral cell sorter, as the mismatch of buffers used for initial sample suspension and the buffers used for sort collection can affect the unmixing of the data, potentially giving rise to erroneous purity check results.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 12","pages":"909-914"},"PeriodicalIF":2.5,"publicationDate":"2024-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24911","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142754754","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":"Barcoding of viable peripheral blood mononuclear cells with selenium and tellurium isotopes for mass cytometry experiments","authors":"Youngran Seo,&nbsp;Ken Fowler,&nbsp;Leah M. Flick,&nbsp;Tracy A. Withers,&nbsp;Barbara Savoldo,&nbsp;Karen McKinnon,&nbsp;Marie A. Iannone","doi":"10.1002/cyto.a.24907","DOIUrl":"10.1002/cyto.a.24907","url":null,"abstract":"<p>Barcoding viable cells combined with pooled sample staining is an effective technique that eliminates batch effects from serial cell staining and facilitates uninterrupted data acquisition. We describe three novel and isotopically pure selenium-containing compounds (SeMals) that are useful cellular labeling tools. The maleimide-functionalized selenophenes (⁷⁶SeMal, ⁷⁷SeMal, and ⁷⁸SeMal) covalently react with cellular sulfhydryl groups and uniquely label cell samples. The SeMal reagents label viable and paraformaldehyde-fixed peripheral blood mononuclear cells (PBMC), are well resolved by the mass cytometer, and have little spill into adjacent channels. They appear non-toxic to viable cells at working concentrations. We used SeMal reagents in combination with four isotopically pure tellurium maleimide reagents (¹²⁴TeMal, ¹²⁶TeMal, ¹²⁸TeMal, and ¹³⁰TeMal) to label 21 individual PBMC samples with unique combinations of selenium and tellurium isotopes (seven donors with three replicates using a 7 isotope pick 2 combinatorial schema). The individually barcoded samples were pooled, stained with an antibody cocktail as a pool, and acquired on the mass cytometer as a single suspension. The single-cell data were de-barcoded into separate sample-specific files after data acquisition, enabling an uninterrupted instrument run. Each donor sample retained its unique phenotypic profile with excellent replicate reproducibility. Unlike current live cell barcoding methods, this approach does not require antibodies to surface markers, allowing for the labeling of all cells regardless of surface antigen expression. Additionally, since selenium and tellurium isotopes are not currently utilized in CyTOF antibody panels, this method expands barcoding options and frees up commonly used isotopes for more detailed cell profiling.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 12","pages":"899-908"},"PeriodicalIF":2.5,"publicationDate":"2024-11-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24907","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142709568","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 11, November 2024 Cover Image","authors":"","doi":"10.1002/cyto.a.24762","DOIUrl":"https://doi.org/10.1002/cyto.a.24762","url":null,"abstract":"","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 11","pages":""},"PeriodicalIF":2.5,"publicationDate":"2024-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24762","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142641790","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":"Cationic lipid transfection induces nuclear actin filaments","authors":"Molika Sinha,&nbsp;Maria Kristha Fernandez,&nbsp;Malte Renz","doi":"10.1002/cyto.a.24903","DOIUrl":"10.1002/cyto.a.24903","url":null,"abstract":"<p>Cationic lipids are widely used for gene delivery. Here, we report the transient formation of nuclear actin filaments in mammalian cells transfected with commercially available transfection reagents regardless of the proteins transfected. Readily detectable with phalloidin, nuclear actin ranges from short filaments to a fully developed network. Nuclear actin filaments persist for hours, peak 20 h after transfection, and may be involved in DNA damage repair.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 12","pages":"893-898"},"PeriodicalIF":2.5,"publicationDate":"2024-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24903","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142615214","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":"Single-cell analysis of osmoregulation reveals heterogeneity of aquaporin 4 functionality in human astrocytes","authors":"Hugo Steenberghen,&nbsp;Sarah De Beuckeleer,&nbsp;Niels Hellings,&nbsp;Veerle Somers,&nbsp;Elise Van Breedam,&nbsp;Peter Ponsaerts,&nbsp;Rony Nuydens,&nbsp;Hervé Maurin,&nbsp;Peter H. Larsen,&nbsp;Winnok H. De Vos","doi":"10.1002/cyto.a.24905","DOIUrl":"10.1002/cyto.a.24905","url":null,"abstract":"<p>The water channel aquaporin 4 (AQP4) contributes to water flow and waste removal across the blood–brain barrier and its levels, organization and localization are perturbed in various neurological diseases, including Alzheimer's Disease. This renders AQP4 a potentially valuable therapeutic target. However, most functional assays aimed at identifying modulators of AQP4 function are performed with primary rodent cells and do not consider inter-cellular variations in AQP4 abundance and presentation. To address this, we have established and applied a robust live cell microscopy assay that captures the contribution of AQP4 in the osmotically driven (de-)quenching of the vital dye Calcein-AM with single-cell resolution. Using human astrocytoma cells, we found that performing measurements on cellular regions instead of whole fields of view yielded a more sensitive readout of the osmotic response, which correlated with AQP4 abundance. Stable co-expression of the two major AQP4 isoforms, but not of the individual isoforms, provoked a faster adaptation to osmotic changes, while siRNA-mediated knockdown of <i>AQP4</i> had the opposite effect. Post-hoc correlation with the canonical membrane marker CD44 revealed that the speed of the osmotic response scaled with AQP4 membrane enrichment. Coating the substrate with laminin promoted AQP4 membrane enrichment, while cell confinement with fixed-size micropatterns further increased the speed of osmoregulation, underscoring the influence of extracellular factors. The osmotic response of primary fetal astrocytes and human iPSC-derived astrocyte models was comparable to AQP4-deficient astrocytoma cells, in line with their low AQP4 levels and indicative of their immature state. In conclusion, a correlative single-cell approach based on the quantification of Calcein-AM quenching capacity, AQP4 abundance and AQP4 membrane enrichment, allows resolving osmoregulation in a more sensitive manner and reveals heterogeneity between and within human astrocyte (–like) cultures, which could prove crucial for future screens aimed at identifying AQP4 modulators.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 12","pages":"870-882"},"PeriodicalIF":2.5,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142582516","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":"Measurable morphological features of single circulating tumor cells in selected solid tumors—A pilot study","authors":"Robert Wenta,&nbsp;Julia Richert,&nbsp;Anna Muchlińska,&nbsp;Elżbieta Senkus,&nbsp;Grażyna Suchodolska,&nbsp;Sylwia Łapińska-Szumczyk,&nbsp;Paweł Domżalski,&nbsp;Kevin Miszewski,&nbsp;Marcin Matuszewski,&nbsp;Rafał Dziadziuszko,&nbsp;Anna Supernat,&nbsp;Anna Żaczek,&nbsp;Natalia Bednarz-Knoll","doi":"10.1002/cyto.a.24906","DOIUrl":"10.1002/cyto.a.24906","url":null,"abstract":"<p>Liquid biopsies developed into a range of sensitive technologies aiming to analyze for example, circulating tumor cells (CTCs) in peripheral blood, which significantly deepens understanding of the metastatic process. Nevertheless, examination of CTCs is mostly limited to their enumeration and usually only 2–3 markers-based phenotyping, not offering yet sufficient insight into their biology. In contrast, quantitative analysis of their morphological details might extend our knowledge about dissemination and even improve CTC isolation or label-free identification methods dependent on their physical features such as size, and deformability. Current study was conducted to describe CTCs' and their size, shape, presence of protrusions, and micronuclei across various types of cancers (lung, <i>n</i> = 29; ovarian, <i>n</i> = 24, breast, <i>n</i> = 54; and prostate, <i>n</i> = 33). Epithelial (pan-keratins), mesenchymal (vimentin), and two exclusion markers were used to identify CTCs and classify them into four epithelial and epithelial-mesenchymal transition-related phenotypes using standardized and throughput method, imaging flow cytometry. The morphological characteristics of CTCs, including their nuclei, such as circularity, the maximum, and minimum diagonal values were determined using an open-source software QuPath. On average, detected CTCs (<i>n</i> = 1156) were larger, and more irregular in shape compared to leukocytes/endothelial cells (<i>n</i> = 400). Epithelial and mesenchymal CTCs had the largest (median = 18.2 μm) and the smallest diameter (median = 10.4 μm), respectively. In terms of cancer-specific variations, the largest CTCs were identified in lung cancer, whereas the smallest—in prostate and breast cancers. Epithelial CTCs and those negative for both epithelial and mesenchymal markers exhibited the highest degree of elongation, whereas mesenchymal CTCs were the most irregular in shape. Protrusions and micronuclei were observed extremely rarely within CTCs of breast and prostate cancer (0.6%–0.8% of CTCs). Micronuclei were observed only in epithelial and epithelial-mesenchymal CTCs. This study underscores the significant variability in the morphological features of CTCs in relation to their phenotypic classification or even the particular organ of origin, potentially influencing for example, size-dependent CTC isolation methods. It demonstrates for the first time the morphological measurements of CTCs undergoing epithelial-mesenchymal transition, and some specific morphological details (i.e., protrusions, micronuclei) within CTCs in general.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 12","pages":"883-892"},"PeriodicalIF":2.5,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24906","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142575113","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 beginner's guide to supervised analysis for mass cytometry data in cancer biology","authors":"Julia Wlosik,&nbsp;Samuel Granjeaud,&nbsp;Laurent Gorvel,&nbsp;Daniel Olive,&nbsp;Anne-Sophie Chretien","doi":"10.1002/cyto.a.24901","DOIUrl":"10.1002/cyto.a.24901","url":null,"abstract":"<p>Mass cytometry enables deep profiling of biological samples at single-cell resolution. This technology is more than relevant in cancer research due to high cellular heterogeneity and complexity. Downstream analysis of high-dimensional datasets increasingly relies on machine learning (ML) to extract clinically relevant information, including supervised algorithms for classification and regression purposes. In cancer research, they are used to develop predictive models that will guide clinical decision making. However, the development of supervised algorithms faces major challenges, such as sufficient validation, before being translated into the clinics. In this work, we provide a framework for the analysis of mass cytometry data with a specific focus on supervised algorithms and practical examples of their applications. We also raise awareness on key issues regarding good practices for researchers curious to implement supervised ML on their mass cytometry data. Finally, we discuss the challenges of supervised ML application to cancer research.</p>","PeriodicalId":11068,"journal":{"name":"Cytometry Part A","volume":"105 12","pages":"853-869"},"PeriodicalIF":2.5,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.24901","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564163","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}