Publications by authors named "John P Nolan"

67 Publications

The evolution of spectral flow cytometry.

Authors:
John P Nolan

Cytometry A 2022 May 14. Epub 2022 May 14.

Scintillon Institute, San Diego, California, USA.

This special issue of Cytometry marks the transition of spectral flow cytometry from an emerging technology into a transformative force that will shape the fields of cytometry and single-cell analysis for some time to come. Tracing its roots to the earliest years of flow cytometry, spectral flow cytometry has evolved from the domain of individual researchers pushing the limits of hardware, reagents, and software to the mainstream, where it is being harnessed and adapted to meet the analytical challenges presented by modern biomedical research. In particular, the current form of spectral flow technology has arisen to address the needs of multiparameter immunophenotyping of immune cells in basic and translational research, and much of the current instrumentation and software reflects the needs of those applications. Yet, the possibilities enabled by high-resolution analysis of the spectral properties of optical absorbance, scatter, and emission have only begun to be exploited. In this brief review, the author highlights the origins and early milestones of single-cell spectral analysis, assesses the current state of instrumentation and software, and speculates as to future directions of spectral flow cytometry technology and applications.
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http://dx.doi.org/10.1002/cyto.a.24566DOI Listing
May 2022

Cancer-cell-secreted miR-122 suppresses O-GlcNAcylation to promote skeletal muscle proteolysis.

Nat Cell Biol 2022 May 25;24(5):793-804. Epub 2022 Apr 25.

Department of Pathology, University of California, San Diego, La Jolla, CA, USA.

A decline in skeletal muscle mass and low muscular strength are prognostic factors in advanced human cancers. Here we found that breast cancer suppressed O-linked N-acetylglucosamine (O-GlcNAc) protein modification in muscle through extracellular-vesicle-encapsulated miR-122, which targets O-GlcNAc transferase (OGT). Mechanistically, O-GlcNAcylation of ryanodine receptor 1 (RYR1) competed with NEK10-mediated phosphorylation and increased K48-linked ubiquitination and proteasomal degradation; the miR-122-mediated decrease in OGT resulted in increased RYR1 abundance. We further found that muscular protein O-GlcNAcylation was regulated by hypoxia and lactate through HIF1A-dependent OGT promoter activation and was elevated after exercise. Suppressed O-GlcNAcylation in the setting of cancer, through increasing RYR1, led to higher cytosolic Ca and calpain protease activation, which triggered cleavage of desmin filaments and myofibrillar destruction. This was associated with reduced skeletal muscle mass and contractility in tumour-bearing mice. Our findings link O-GlcNAcylation to muscular protein homoeostasis and contractility and reveal a mechanism of cancer-associated muscle dysregulation.
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http://dx.doi.org/10.1038/s41556-022-00893-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9107513PMC
May 2022

S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia.

J Neurosci 2022 04 15;42(14):3011-3024. Epub 2022 Feb 15.

Neurodegeneration New Medicines Center and Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037

Dysregulation of autophagic pathways leads to accumulation of abnormal proteins and damaged organelles in many neurodegenerative disorders, including Parkinson's disease (PD) and Lewy body dementia (LBD). Autophagy-related dysfunction may also trigger secretion and spread of misfolded proteins, such as α-synuclein (α-syn), the major misfolded protein found in PD/LBD. However, the mechanism underlying these phenomena remains largely unknown. Here, we used cell-based models, including human induced pluripotent stem cell-derived neurons, CRISPR/Cas9 technology, and male transgenic PD/LBD mice, plus vetting in human postmortem brains (both male and female). We provide mechanistic insight into this pathologic pathway. We find that aberrant S-nitrosylation of the autophagic adaptor protein p62 causes inhibition of autophagic flux and intracellular buildup of misfolded proteins, with consequent secretion resulting in cell-to-cell spread. Thus, our data show that pathologic protein S-nitrosylation of p62 represents a critical factor not only for autophagic inhibition and demise of individual neurons, but also for α-syn release and spread of disease throughout the nervous system. In Parkinson's disease and Lewy body dementia, dysfunctional autophagy contributes to accumulation and spread of aggregated α-synuclein. Here, we provide evidence that protein S-nitrosylation of p62 inhibits autophagic flux, contributing to α-synuclein aggregation and spread.
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http://dx.doi.org/10.1523/JNEUROSCI.1508-21.2022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8985870PMC
April 2022

Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release.

Front Pharmacol 2021 16;12:668609. Epub 2021 Apr 16.

Moores Cancer Center, University of California, San Diego, La Jolla, CA, United States.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z' factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.
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http://dx.doi.org/10.3389/fphar.2021.668609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085554PMC
April 2021

Methamphetamine use alters human plasma extracellular vesicles and their microRNA cargo: An exploratory study.

J Extracell Vesicles 2020 11 28;10(1):e12028. Epub 2020 Nov 28.

Department of Anesthesiology & Perioperative Medicine Oregon Health & Science University Portland Oregon USA.

Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neurotoxicity and cardiovascular disease. Recent studies have begun to link microRNAs (miRNAs) to the processes related to MA use and addiction. Our studies are the first to analyse plasma EVs and their miRNA cargo in humans actively using MA (MA-ACT) and control participants (CTL). In this cohort we also assessed the effects of tobacco use on plasma EVs. We used vesicle flow cytometry to show that the MA-ACT group had an increased abundance of EV tetraspanin markers (CD9, CD63, CD81), but not pro-coagulant, platelet-, and red blood cell-derived EVs. We also found that of the 169 plasma EV miRNAs, eight were of interest in MA-ACT based on multiple statistical criteria. In smokers, we identified 15 miRNAs of interest, two that overlapped with the eight MA-ACT miRNAs. Three of the MA-ACT miRNAs significantly correlated with clinical features of MA use and target prediction with these miRNAs identified pathways implicated in MA use, including cardiovascular disease and neuroinflammation. Together our findings indicate that MA use regulates EVs and their miRNA cargo, and support that further studies are warranted to investigate their mechanistic role in addiction, recovery, and recidivism.
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http://dx.doi.org/10.1002/jev2.12028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890470PMC
November 2020

Psychometric Properties of the Assessment of Perceived Access to Care (APAC) Instrument.

J Ambul Care Manage 2021 Jan/Mar;44(1):31-45

Department of Psychiatry, School of Medicine, University of Washington, Seattle (Dr Fortney and Hawrilenko and Mr Bechtel); Department of Veterans Affairs, Health Services Research and Development, Center of Innovation for Veteran-Centered and Value-Driven Care, Seattle, Washington (Dr Fortney); Department of Psychiatry, College of Medicine, University of Arkansas for Medical Sciences, Little Rock (Dr Pyne); Department of Veterans Affairs, Health Services Research and Development, Center for Mental Healthcare and Outcomes Research, Little Rock, Arkansas (Dr Pyne); Social and Economic Sciences Research Center at Washington State University, Pullman (Dr Moore); University of Michigan Medical School, Ann Arbor (Dr Pfeiffer); Department of Veterans Affairs, Health Services Research and Development, Center for Clinical Management Research, VA Ann Arbor Healthcare System, Ann Arbor, Michigan (Dr Pfeiffer); Community Health Plan of Washington, Seattle (Ms Shushan); Department of Psychiatry, University of Colorado at Denver (Dr Shore); and Department of Bioethics and Humanities, School of Medicine, University of Washington, Seattle (Dr Bowen). Mr Nolan is Veteran Advocate and a member of the SPIRIT Consumer Advisory Board.

Valid measures of perceived access are needed to measure whether health care systems are providing adequate access. This research reports on the psychometric properties of the Assessment of Perceived Access to Care (APAC), which was administered to 1004 Community Health Center patients screening positive for psychiatric disorders. Known-group validity was good, with 6 of the 8 hypothesized associations between social determinants of access and perceived access being significant (P < .01). Better access was significantly (P < .01) correlated with more outpatient mental health visits, indicating good convergent validity. The test-retest Pearson correlation coefficient (0.64) was statistically significant (P < .01). The APAC has acceptable psychometric properties.
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http://dx.doi.org/10.1097/JAC.0000000000000358DOI Listing
October 2021

Aiming to Compare Apples to Apples: Analysis of Extracellular Vesicles and Other Nanosized Particles by Flow Cytometry.

Cytometry A 2020 06;97(6):566-568

Scintillon Institute, San Diego, California, USA.

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http://dx.doi.org/10.1002/cyto.a.24173DOI Listing
June 2020

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

J Extracell Vesicles 2020 3;9(1):1713526. Epub 2020 Feb 3.

Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
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http://dx.doi.org/10.1080/20013078.2020.1713526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034442PMC
February 2020

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

Methodology for evaluating and comparing flow cytometers: A multisite study of 23 instruments.

Cytometry A 2018 11 23;93(11):1087-1091. Epub 2018 Sep 23.

Wake Forest University Baptist Medical Center, Comprehensive Cancer Center and Department of Cancer Biology, Winston-Salem, North Carolina.

We demonstrate improved methods for making valid and accurate comparisons of fluorescence measurement capabilities among instruments tested at different sites and times. We designed a suite of measurements and automated data processing methods to obtain consistent objective results and applied them to a selection of 23 instruments at nine sites to provide a range of instruments as well as multiple instances of similar instruments. As far as we know, this study represents the most accurate methods and results so far demonstrated for this purpose. The first component of the study reporting improved methods for photoelectron scale (Spe) evaluations, which was published previously (Parks, El Khettabi, Chase, Hoffman, Perfetto, Spidlen, Wood, Moore, and Brinkman: Cytometry A 91 (2017) 232-249). Those results which were within themselves are not sufficient for instrument comparisons, so here, we use the Spe scale results for the 23 cytometers and combine them with additional information from the analysis suite to obtain the metrics actually needed for instrument evaluations and comparisons. We adopted what we call the 2+2SD limit of resolution as a maximally informative metric, for evaluating and comparing dye measurement sensitivity among different instruments and measurement channels. Our results demonstrate substantial differences among different classes of instruments in both dye response and detection sensitivity and some surprisingly large differences among similar instruments, even among instruments with nominally identical configurations. On some instruments, we detected defective measurement channels needing service. The system can be applied in shared resource laboratories and other facilities as an aspect of quality assurance, and accurate instrument comparisons can be valuable for selecting instruments for particular purposes and for making informed instrument acquisition decisions. An institutionally supported program could serve the cytometry community by facilitating access to materials, and analysis and maintaining an archive of results. © 2018 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901711PMC
November 2018

Investigation into the Mechanism(s) That Leads to Platelet Decreases in Cynomolgus Monkeys During Administration of ISIS 104838, a 2'-MOE-Modified Antisense Oligonucleotide.

Toxicol Sci 2018 08;164(2):613-626

Nonclinical Development, Ionis Pharmaceuticals Inc, Carlsbad, California, 92010.

ISIS 104838, a 2'-O-methoxyethyl (2'-MOE)-modified antisense oligonucleotide (ASO), causes a moderate, reproducible, dose-dependent, but selflimiting decrease in platelet (PLT) counts in monkeys and humans. To determine the etiology of PLT decrease in cynomolgus monkeys, a 12-week repeat dose toxicology study in 5 cynomolgus monkeys given subcutaneous injections of ISIS 104838 (30-60 mg/kg/week). Monkeys were also injected intravenously with 111Indium(In)-oxine-labeled PLTs to investigate PLT sequestration. In response to continued dosing, PLT counts were decreased by 50%-90% by day 30 in all monkeys. PLT decreases were accompanied by 2- to 4.5-fold increases in immunoglobulin M(IgM), which were typified by a 2- to 5-fold increase in antiplatelet factor 4 (antiPF4) IgM and antiPLT IgM, respectively. Monocyte chemotactic protein 1 increased upon dosing of ISIS 104838, concomitant with a 2- to 6-fold increase in monocyte-derived extracellular vesicles (EVs), indicating monocyte activation but not PLT activation. Despite a 2- to 3-fold increase in von Willebrand factor antigen in all monkeys following ASO administration, only 2 monkeys showed a 2- to 4-fold increase in endothelial EVs. Additionally, a ∼60 - 80%% increase in PLT sequestration in liver and spleen was also observed. Collectively, these results suggest the overall increase in total IgM, antiPLT IgM and/or antiPF4 IgM, in concert with monocyte activation contributed to increased PLT sequestration in spleen and liver, leading to decreased PLTs in peripheral blood.
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http://dx.doi.org/10.1093/toxsci/kfy119DOI Listing
August 2018

Advances, challenges, and opportunities in extracellular RNA biology: insights from the NIH exRNA Strategic Workshop.

JCI Insight 2018 04 5;3(7). Epub 2018 Apr 5.

Cancer Immunology, Hematology, and Etiology Branch, Division of Cancer Biology, National Cancer Institute, Bethesda, Maryland, USA.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.
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http://dx.doi.org/10.1172/jci.insight.98942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928855PMC
April 2018

Analysis of Individual Extracellular Vesicles by Flow Cytometry.

Methods Mol Biol 2018 ;1678:79-92

Scintillon Institute, San Diego, CA, 92121, USA.

Extracellular vesicles (EVs) are released by cells and can be found in cell culture supernatants and biofluids. EVs carry proteins, nucleic acids, and other cellular components and can deliver these to nearby or distant cells, making EVs of interest as both disease biomarkers and therapeutic targets. EVs in biofluids are heterogeneous, coming from different cell types and from different sources with the cell, which limits the usefulness of bulk EV analysis methods that report the average features of all EVs present. Single-particle measurements such as flow cytometry would be preferred, but the small size and low abundance of surface antigens challenges conventional flow cytometry approaches, leading to the development of vesicle-specific assays and experimental design. Among the key issues that have emerged are: (a) judicious choice of detection (triggering) approach; (b) appropriate control experiments to confirm the vesicular nature of the detected events and the contribution of coincidence (aka swarm detection); and (c) the importance of fluorescence calibration to allow data to be compared over time and between laboratories. We illustrate these issues in the context of fluorescence-triggered Vesicle Flow Cytometry (VFC), a general approach to the quantitative measurement of EV number, size, and surface marker expression.
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http://dx.doi.org/10.1007/978-1-4939-7346-0_5DOI Listing
June 2018

Analysis of extracellular RNA in cerebrospinal fluid.

J Extracell Vesicles 2017 24;6(1):1317577. Epub 2017 May 24.

Department of Neurosurgery, University of California, San Diego, CA, USA.

We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer's disease (AD), Parkinson's disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained ~106 EVs/μL by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.
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http://dx.doi.org/10.1080/20013078.2017.1317577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505019PMC
May 2017

Methodological Guidelines to Study Extracellular Vesicles.

Circ Res 2017 May;120(10):1632-1648

From the Biomedical Engineering and Physics (F.A.W.C., T.G.v.L., E.v.d.P.), Vesicle Observation Centre (F.A.W.C., A.G., T.G.v.L., E.v.d.P., G.S., R.N.), and Laboratory of Experimental Clinical Chemistry (A.G., G.S., R.N.), Academic Medical Center, University of Amsterdam, The Netherlands; Extracellular Vesicles and Membrane Repair, UMR-5248-CBMN CNRS, University of Bordeaux, IPB, Pessac, France (A.R.B.); Department of Genetics, Cell- and Immunobology, Semmelweis University, Budapest, Hungary (E.I.B.); VRCM, UMRS-1076, INSERM, Aix-Marseille University, UFR de Pharmacie, Marseille, France (F.D.-G., R.L.); Haematology and vascular biology department, CHU La Conception, APHM, Marseille, France (F.D.-G., R.L.); Exosomes Research Group, Department of Pathology, VU University Medical Center, De Boelelaan 1117, Amsterdam, The Netherlands (E.E.E.D., D.M.P.); Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden (S.E.-A., Y.L.); Department of Physiology, Anatomy and Genetics, University of Oxford, United Kingdom (S.E.-A., I.M.); Bristol Heart Institute, University of Bristol, United Kingdom (C.E.); National Heart & Lung Institute, Imperial College London, United Kingdom (C.E.); 1st Chair and Department of Cardiology, Medical University of Warsaw, Poland (A.G.); Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Belgium (A.H., O.d.W.); Cancer Research Institute Ghent, Belgium (A.H., O.d.W.); Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia (A.F.H.); Department of Medicine, University of North Carolina at Chapel Hill (N.M.); Institute of Technology, University of Tartu, Estonia (I.M.); Scintillon Institute, San Diego, CA (J.P.N.); Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY (S.S.); and EV Core Facility, University of Helsinki and EV-Group, Division of Biochemistry and Biotechnology, Department of Biosciences, University of Helsinki, Finland (P.R.M.S.).

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.309417DOI Listing
May 2017

Detection of platelet vesicles by flow cytometry.

Platelets 2017 May 2;28(3):256-262. Epub 2017 Mar 2.

b Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda , MD , USA.

The composition and function of platelet-derived extracellular vesicles (EVs) in health and in disease are a major topic of investigation in biomedical research. However, efforts to delineate specific molecular repertoires and roles for different types of EVs in the circulation are limited not only by the lack of flow cytometers capable of analyzing submicron- and nano-materials across the full size spectrum of plasma EVs, but also by the lack of standardized methods and reference materials that would permit inter-laboratory reproducibility for these analyses. In this review, we summarize the flow cytometry of EVs, with a focus on platelet vesicles in plasma. In addition to delineating the basic principles that govern what precautions must be considered when using flow cytometry for the analysis of platelet vesicles, we provide an overview for how to standardize, control, annotate, and report EV flow cytometry data reproducibly, while looking forward to a next generation of high sensitivity instruments for the analysis of EVs and other submicron biomaterials in the circulation.
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http://dx.doi.org/10.1080/09537104.2017.1280602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415413PMC
May 2017

Non-Lethal Endotoxin Injection: A Rat Model of Hypercoagulability.

PLoS One 2017 12;12(1):e0169976. Epub 2017 Jan 12.

Pathology Department, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, United States of America.

Systemic inflammation co-activates coagulation, which unchecked culminates in a lethal syndrome of multi-organ microvascular thrombosis known as disseminated intravascular coagulation (DIC). We studied an endotoxin-induced inflammatory state in rats to identify biomarkers of hemostatic imbalance favoring hypercoagulability. Intraperitoneal injection of LPS at 15 mg/kg body weight resulted in peripheral leukopenia and widespread neutrophilic sequestration characteristic of an acute systemic inflammatory response. Early indicators of hemostatic pathway activation developed within 4 hours, including increased circulating concentrations of procoagulant extracellular vesicles (EVs), EVs expressing endothelial cell and platelet membrane markers, and high concentration of soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor-1 (PAI-1), and D-dimers. Inflammation persisted throughout the 48-hour observation period; however, increases were found in a subset of serum microRNA (miRNA) that coincided with gradual resolution of hemostatic protein abnormalities and reduction in EV counts. Dose-adjusted LPS treatment in rats provides a time-course model to develop biomarker profiles reflecting procoagulant imbalance and rebalance under inflammatory conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169976PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233421PMC
August 2017

Extracellular vesicles: Great potential, many challenges.

Cytometry B Clin Cytom 2016 07 24;90(4):324-5. Epub 2016 May 24.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine of the University of Pennsylvania, Philadelphia, Pennsylvania.

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http://dx.doi.org/10.1002/cyto.b.21375DOI Listing
July 2016

Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF).

PLoS One 2016 22;11(2):e0149866. Epub 2016 Feb 22.

Center for Theoretical and Applied Neuro-Oncology, University of California San Diego, San Diego, California, United States of America.

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149866PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763994PMC
July 2016

Measurement of extracellular vesicles and other submicron size particles by flow cytometry.

Cytometry A 2016 Feb;89(2):109-10

National Health and Environmental Effects Research Laboratory, Office of Research and Development, Toxicology Assessment Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina.

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http://dx.doi.org/10.1002/cyto.a.22814DOI Listing
February 2016

High sensitivity flow cytometry of membrane vesicles.

Cytometry A 2016 Feb 20;89(2):196-206. Epub 2015 Oct 20.

La Jolla Bioengineering Institute, La Jolla, California, 92037.

Extracellular vesicles (EVs) are attracting attention as vehicles for inter-cellular signaling that may have value as diagnostic or therapeutic targets. EVs are released by many cell types and by different mechanisms, resulting in phenotypic heterogeneity that makes them a challenge to study. Flow cytometry is a popular tool for characterizing heterogeneous mixtures of particles such as cell types within blood, but the small size of EVs makes them difficult to measure using conventional flow cytometry. To address this limitation, a high sensitivity flow cytometer was constructed and EV measurement approaches that allowed them to enumerate and estimate the size of individual EVs, as well as measure the presence of surface markers to identify phenotypic subsets of EVs. Several fluorescent membrane probes were evaluated and it was found that the voltage sensing dye di-8-ANEPPS could produce vesicle fluorescence in proportion to vesicle surface area, allowing for accurate measurements of EV number and size. Fluorescence-labeled annexin V and anti-CD61 antibody was used to measure the abundance of these surface markers on EVs in rat plasma. It was shown that treatment of platelet rich plasma with calcium ionophore resulted in an increase in the fraction of annexin V and CD61-positive EVs. Vesicle flow cytometry using fluorescence-based detection of EVs has the potential to realize the potential of cell-derived membrane vesicles as functional biomarkers for a variety of applications.
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http://dx.doi.org/10.1002/cyto.a.22787DOI Listing
February 2016

Multiplexed TEM Specimen Preparation and Analysis of Plasmonic Nanoparticles.

Microsc Microanal 2015 Aug;21(4):1017-1025

The National Resource for Automated Molecular Microscopy, La Jolla, California, 92037, USA.

We describe a system for rapidly screening hundreds of nanoparticle samples using transmission electron microscopy (TEM). The system uses a liquid handling robot to place up to 96 individual samples onto a single standard TEM grid at separate locations. The grid is then transferred into the TEM and automated software is used to acquire multiscale images of each sample. The images are then analyzed to extract metrics on the size, shape, and morphology of the nanoparticles. The system has been used to characterize plasmonically active nanomaterials.
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http://dx.doi.org/10.1017/S1431927615014324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701052PMC
August 2015

Flow Cytometry of Extracellular Vesicles: Potential, Pitfalls, and Prospects.

Authors:
John P Nolan

Curr Protoc Cytom 2015 Jul 1;73:13.14.1-13.14.16. Epub 2015 Jul 1.

Scintillon Institute, San Diego, California.

Evidence suggests that extracellular vesicles (EVs) can play roles in physiology and pathology, providing impetus to explore their use as diagnostic and therapeutic targets. However, EVs are also small, heterogeneous, and difficult to measure, and so this potential has not yet been realized. The development of improved approaches to EV detection and characterization will be critical to further understanding their roles in physiology and disease. Flow cytometry has been a popular tool for measuring cell-derived EVs, but has often been used in an uncritical manner in which fundamental principles and limitations of the instrument are ignored. Recent efforts to standardize procedures and document the effects of different methodologies have helped to address this shortcoming, but much work remains. In this paper, I address some of the instrument, reagent, and analysis considerations relevant to measurement of individual EVs in flow, with the aim of clarifying a path to quantitative and standardized measurement of these interesting and potentially important biological nanoparticles.
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http://dx.doi.org/10.1002/0471142956.cy1314s73DOI Listing
July 2015

Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles.

ACS Nano 2014 Oct 13;8(10):10998-1006. Epub 2014 Oct 13.

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University , Xiamen, Fujian 361005, People's Republic of China.

Ultrasensitive detection and characterization of single nanoparticles (<100 nm) is important in nanotechnology and life sciences. Direct measurement of the elastically scattered light from individual nanoparticles represents the simplest and the most direct method for particle detection. However, the sixth-power dependence of scattering intensity on particle size renders very small particles indistinguishable from the background. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, here we report the development of high sensitivity flow cytometry (HSFCM) that achieves real-time light-scattering detection of single silica and gold nanoparticles as small as 24 and 7 nm in diameter, respectively. This unprecedented sensitivity enables high-resolution sizing of single nanoparticles directly based on their scattered intensity. With a resolution comparable to that of TEM and the ease and speed of flow cytometric analysis, HSFCM is particularly suitable for nanoparticle size distribution analysis of polydisperse/heterogeneous/mixed samples. Through concurrent fluorescence detection, simultaneous insights into the size and payload variations of engineered nanoparticles are demonstrated with two forms of clinical nanomedicine. By offering quantitative multiparameter analysis of single nanoparticles in liquid suspensions at a throughput of up to 10 000 particles per minute, HSFCM represents a major advance both in light-scattering detection technology and in nanoparticle characterization.
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http://dx.doi.org/10.1021/nn505162uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4212780PMC
October 2014

Optimization of SERS tag intensity, binding footprint, and emittance.

Bioconjug Chem 2014 Jul 10;25(7):1233-42. Epub 2014 Jun 10.

La Jolla Bioengineering Institute Suite 210 3535 General Atomics Court San Diego, California 92121, United States.

Nanoparticle surface enhanced Raman scattering (SERS) tags have attracted interest as labels for use in a variety of applications, including biomolecular assays. An obstacle to progress in this area is a lack of standardized approaches to compare the brightness of different SERS tags within and between laboratories. Here we present an approach based on binding of SERS tags to beads with known binding capacities that allows evaluation of the average intensity, the relative binding footprint of particles in a SERS tag preparation, and the size-normalized intensity or emittance. We tested this on four different SERS tag compositions and show that aggregated gold nanorods produce SERS tags that are 2-4 times brighter than relatively more monodisperse nanorods, but that the aggregated nanorods are also correspondingly larger, which may negate the intensity if steric hindrance limits the number of tags bound to a target. By contrast, SERS tags prepared from smaller gold nanorods coated with a silver shell produce SERS tags that are 2-3 times brighter, on a size-normalized basis, than the Au nanorod-based tags, resulting in labels with improved performance in SERS-based image and flow cytometry assays. SERS tags based on red-resonant Ag plates showed similarly bright signals and small footprint. This approach to evaluating SERS tag brightness is general, uses readily available reagents and instruments, and should be suitable for interlab comparisons of SERS tag brightness.
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http://dx.doi.org/10.1021/bc5000252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215889PMC
July 2014

Circulating tumor cell identification by functionalized silver-gold nanorods with multicolor, super-enhanced SERS and photothermal resonances.

Sci Rep 2014 May 9;4:4752. Epub 2014 May 9.

Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, 2801 S. University Avenue, Little Rock, Arkansas 72204, USA.

Nanotechnology has been extensively explored for cancer diagnostics. However, the specificity of current methods to identify simultaneously several cancer biomarkers is limited due to color overlapping of bio-conjugated nanoparticles. Here, we present a technique to increase both the molecular and spectral specificity of cancer diagnosis by using tunable silver-gold nanorods with narrow surface-enhanced Raman scattering (SERS) and high photothermal contrast. The silver-gold nanorods were functionalized with four Raman-active molecules and four antibodies specific to breast cancer markers and with leukocyte-specific CD45 marker. More than two orders of magnitude of SERS signal enhancement was observed from these hybrid nanosystems compared to conventional gold nanorods. Using an antibody rainbow cocktail, we demonstrated highly specific detection of single breast cancer cells in unprocessed human blood. By integrating multiplex targeting, multicolor coding, and multimodal detection, our approach has the potential to improve multispectral imaging of individual tumor cells in complex biological environments.
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http://dx.doi.org/10.1038/srep04752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015134PMC
May 2014

Knowing the code.

Authors:
John P Nolan

Cytometry A 2014 Jan;85(1):10-1

La Jolla Bioengineering Institute, La Jolla, California.

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http://dx.doi.org/10.1002/cyto.a.22419DOI Listing
January 2014

A trigger channel threshold artifact in nanoparticle analysis.

Cytometry A 2013 Mar 18;83(3):301-5. Epub 2013 Jan 18.

La Jolla Bioengineering Institute, San Diego, California 92037, USA.

The analysis of individual nanoparticles by flow cytometry involves the measurement of dim signals that are near the detection limits of the instrument. Discriminating the signal from particles of interest from that of background particles in buffers and from optical and electronic noise can be challenging, and requires careful consideration of the measurement approach, control experiments, and scrutiny of the resulting data. In applying this scrutiny, we have come to recognize an artifact that results from the inappropriate selection of the trigger channel threshold that might not be obvious to the casual user. When measuring dim signals close to the noise or background levels, it is intuitive and common for the operator to adjust the trigger threshold to minimize the "false triggers" acquired by the system, and then to run the unknown sample, interpreting the events detected above the background as measurements of individual particles. We show here that when this approach is used to measure particles whose signals fall below the trigger threshold, only coincident events are detected, producing erroneous measurements of both particle number and brightness. We suggest that in many cases, the analysis of dim nanoparticles is best achieved using a fluorescence channel for the trigger.
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http://dx.doi.org/10.1002/cyto.a.22255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578978PMC
March 2013

Spectral flow cytometry.

Curr Protoc Cytom 2013 Jan;Chapter 1:Unit1.27

La Jolla Bioengineering Institute, San Diego, California, USA.

Interest in measuring the complete fluorescence spectra of individual cells in flow can be traced to the earliest days of flow cytometry. Recent advances in detectors, optics, and computation have made it possible to make full spectral measurements in the sub-millisecond time frame in which flow cytometry measurements typically occur. This opens up new possibilities for applying spectroscopy to the analysis of individual cells. This unit reviews historical and contemporary approaches to spectral flow cytometry, as well as instrument design, calibration, and data analysis for spectral flow cytometry applications.
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http://dx.doi.org/10.1002/0471142956.cy0127s63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556726PMC
January 2013

Visible and near infrared fluorescence spectral flow cytometry.

Cytometry A 2013 Mar 6;83(3):253-64. Epub 2012 Dec 6.

La Jolla Bioengineering Institute, San Diego, California, USA.

There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection. Customized software allows the single cell spectral data to be displayed and analyzed to produce new spectra-derived parameters. We show that familiar reference and calibration beads can be employed to quantitatively assess instrument performance. We use microspheres stained with six different quantum dots to compare a virtual bandpass filter approach with classic least squares (CLS) spectral unmixing, and then use antibody capture beads and CLS unmixing to demonstrate immunophenotyping of peripheral blood mononuclear cells using spectral flow cytometry. Finally, we characterize and evaluate several near infrared (NIR) emitting fluorophores for use in spectral flow cytometry. Spectral flow cytometry offers a number of attractive features for single cell analysis, including a simplified optical path, high spectral resolution, and streamlined approaches to quantitative multiparameter measurements. The availability of robust instrumentation, software, and analysis approaches will facilitate the development of spectral flow cytometry applications.
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http://dx.doi.org/10.1002/cyto.a.22241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594514PMC
March 2013
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