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2.
Nat Methods ; 18(8): 903-911, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34354295

RESUMO

The development of DNA-barcoded antibodies to tag cell surface molecules has enabled the use of droplet-based single-cell sequencing (dsc-seq) to profile protein abundances from thousands of cells simultaneously. As compared to flow and mass cytometry, the high per cell cost of current dsc-seq-based workflows precludes their use in clinical applications and large-scale pooled screens. Here, we introduce SCITO-seq, a workflow that uses splint oligonucleotides (oligos) to enable combinatorially indexed dsc-seq of DNA-barcoded antibodies from over 105 cells per reaction using commercial microfluidics. By encoding sample barcodes into splint oligos, we demonstrate that multiplexed SCITO-seq produces reproducible estimates of cellular composition and surface protein expression comparable to those from mass cytometry. We further demonstrate two modified splint oligo designs that extend SCITO-seq to achieve compatibility with commercial DNA-barcoded antibodies and simultaneous expression profiling of the transcriptome and surface proteins from the same cell. These results demonstrate SCITO-seq as a flexible and ultra-high-throughput platform for sequencing-based single-cell protein and multimodal profiling.


Assuntos
Citometria de Fluxo/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microfluídica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Humanos
3.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361020

RESUMO

A stochastic model of the flow cytometer measurement process was developed to assess the nature of the observed coefficient of variation (CV%) of the mean fluorescence intensity (MFI) from a population of labeled microspheres (beads). Several sources of variability were considered: the total number of labels on a bead, the path through the laser beam, the optical absorption cross-section, the quantum yield, the numerical aperture of the collection optics, and the photoelectron conversion efficiency of the photomultiplier (PMT) cathode. The variation in the number of labels on a bead had the largest effect on the CV% of the MFI of the bead population. The variation in the path of the bead through the laser beam was minimized using flat-top lasers. The variability in the average optical properties of the labels was of minor importance for beads with sufficiently large number of labels. The application of the bead results to the measured CV% of labeled B cells indicated that the measured CV% was a reliable measure of the variability of antibodies bound per cell. With some modifications, the model can be extended to multicolor flow cytometers and to the study of CV% from cells with low fluorescence signal.


Assuntos
Linfócitos B/citologia , Citometria de Fluxo/normas , Microesferas , Análise de Variância , Citometria de Fluxo/métodos , Humanos , Reprodutibilidade dos Testes , Processos Estocásticos
4.
Int J Lab Hematol ; 43 Suppl 1: 54-64, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34288436

RESUMO

Ever since hematopoietic cells became "events" enumerated and characterized in suspension by cell counters or flow cytometers, researchers and engineers have strived to refine the acquisition and display of the electronic signals generated. A large array of solutions was then developed to identify at best the numerous cell subsets that can be delineated, notably among hematopoietic cells. As instruments became more and more stable and robust, the focus moved to analytic software. Almost concomitantly, the capacity increased to use large panels (both with mass and classical cytometry) and to apply artificial intelligence/machine learning for their analysis. The combination of these concepts raised new analytical possibilities, opening an unprecedented field of subtle exploration for many conditions, including hematopoiesis and hematological disorders. In this review, the general concepts and progress achieved in the development of new analytical approaches for exploring high-dimensional data sets at the single-cell level will be described as they appeared over the past few years. A larger and more practical part will detail the various steps that need to be mastered, both in data acquisition and in the preanalytical check of data files. Finally, a step-by-step explanation of the solution in development to combine the Bioconductor clustering algorithm FlowSOM and the popular and widely used software Kaluza® (Beckman Coulter) will be presented. The aim of this review was to point out that the day when these progresses will reach routine hematology laboratories does not seem so far away.


Assuntos
Citometria de Fluxo/métodos , Neoplasias Hematológicas/diagnóstico , Aprendizado de Máquina não Supervisionado , Algoritmos , Medula Óssea/metabolismo , Medula Óssea/patologia , Análise por Conglomerados , Biologia Computacional/métodos , Progressão da Doença , Neoplasias Hematológicas/etiologia , Humanos , Modelos Teóricos , Software
5.
Int J Lab Hematol ; 43 Suppl 1: 71-77, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34288444

RESUMO

Therapies in myeloma are rapidly advancing with a host of new targeted therapies coming to market. While these drugs offer significant survival benefits and better side-effect profiles compared with conventional chemotherapeutics, they raise significant difficulties in monitoring post-therapy disease status by flow cytometry due to assay interference and/or selection of phenotypically different sub-clones. The principal culprit, anti-CD38 monoclonal antibodies, limits the ability to detect plasma cells based on classical CD38/CD45 gating. Other markers, such as CD138, are known to be suboptimal by flow cytometry. Various techniques have been proposed to overcome this problem. The most promising of these techniques has been the marker VS38c, a monoclonal antibody targeting an endoplasmic reticulum protein which has shown high sensitivity for plasma cells. Alternative techniques for gating plasma cells, while variably effective in the near term are already the subject of several targeted therapies rendering their usefulness limited in the longer term. Likewise, future targets of these therapies may render present aberrancy markers ineffective in MRD testing. These therapies pose challenges that must be overcome with new markers and novel panels in order for flow cytometric MRD testing to remain relevant.


Assuntos
Citometria de Fluxo , Mieloma Múltiplo/diagnóstico , Neoplasia Residual/diagnóstico , Gerenciamento Clínico , Suscetibilidade a Doenças , Citometria de Fluxo/métodos , Humanos , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/etiologia , Neoplasia Residual/tratamento farmacológico
6.
Int J Lab Hematol ; 43 Suppl 1: 43-53, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34288449

RESUMO

Multiple myeloma (MM) is a heterogeneous group of mature B-cell diseases that are typically characterized by the presence and accumulation of abnormal plasma cells (PCs), which results in the excess production of monoclonal immunoglobulin and/or light chain found in the serum and/or urine. Multiparametric flow cytometry (MFC) is an indispensable tool to supplement the diagnosis, classification and monitoring of the disease due to its high patient applicability, excellent sensitivity and encouraging results from various clinical trials. In this regard, minimal or, more appropriately, measurable residual disease (MRD) negativity by MFC has been recognized as a powerful predictor of favourable long-term outcomes. Before flow cytometry can be effectively implemented in the clinical setting for MM MRD testing, sample preparation, panel configuration, analysis and gating strategies must be optimized to ensure accurate results. This manuscript will discuss the current consensus guidelines for flow cytometric processing of samples and reporting of results for MM MRD testing. We also discuss alternative approaches to detect plasma cells in the presence of daratumumab treatment. Finally, there is a lack of information describing the subclonal distribution of myeloma cells based on their protein expression. The advent of high-dimensional analysis may assist in following the evolution of antigen expression patterns on abnormal plasma cells in patients with relapsed/refractory disease. This in turn can help identify clonal subtypes that are more aggressive for potential informed decision. An analysis using t-SNE to identify the emergence of PCs subclones by MFC, along with the analysis of their immunophenotypic profiles are presented as a future perspective.


Assuntos
Citometria de Fluxo , Imunofenotipagem , Mieloma Múltiplo/diagnóstico , Neoplasia Residual/diagnóstico , Biomarcadores Tumorais , Análise de Dados , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Imunofenotipagem/métodos , Imunofenotipagem/normas , Guias de Prática Clínica como Assunto , Reprodutibilidade dos Testes , Projetos de Pesquisa , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Manejo de Espécimes/normas
7.
Methods Mol Biol ; 2289: 47-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270062

RESUMO

Efficient doubled haploid (DH) plant production is of great interest in the plant breeding industry and research because homozygous lines are obtained within a single generation shortening the breeding cycle substantially. DH protocol development can be a time- and resource-consuming process due to numerous factors affecting its success and efficiency. Here we present concepts and examples about how critical success factors can be identified throughout a DH protocol and an early microspore response monitored by simple impedance flow cytometry (IFC) measurements, which will help to optimize each step of an androgenesis-based DH protocol.


Assuntos
Citometria de Fluxo/métodos , Tecnologia/métodos , Impedância Elétrica , Haploidia , Melhoramento Vegetal/métodos , Plantas/genética
8.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299223

RESUMO

Seaweeds are one of the largest producers of biomass in the marine environment and a source of multiple bioactive metabolites with valuable health benefits. Among these, phlorotannins have been widely recognized for their promising bioactive properties. The potential antitumor capacity of Fucus vesiculosus-derived phlorotannins remains, however, poorly explored, especially in gastrointestinal tract-related tumors. Therefore, this work aimed to evaluate the cytotoxic properties and possible mechanisms by which F. vesiculosus crude extract (CRD), phlorotannin-rich extract (EtOAc), and further phlorotannin-purified fractions (F1-F9) trigger cell death on different tumor cell lines of the gastrointestinal tract, using flow cytometry. The results indicate that F. vesiculosus samples exert specific cytotoxicity against tumor cell lines without affecting the viability of normal cells. Moreover, it was found that, among the nine different phlorotannin fractions tested, F5 was the most active against both Caco-2 colorectal and MKN-28 gastric cancer cells, inducing death via activation of both apoptosis and necrosis. The UHPLC-MS analysis of this fraction revealed, among others, the presence of a compound tentatively identified as eckstolonol and another as fucofurodiphlorethol, which could be mainly responsible for the promising cytotoxic effects observed in this sample. Overall, the results herein reported contribute to a better understanding of the mechanisms behind the antitumor properties of F. vesiculosus phlorotannin-rich extracts.


Assuntos
Fucus/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Taninos/farmacologia , Apoptose/efeitos dos fármacos , Células CACO-2 , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Citometria de Fluxo/métodos , Humanos , Extratos Vegetais/farmacologia , Alga Marinha/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo
9.
EBioMedicine ; 69: 103464, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34233260

RESUMO

BACKGROUND: The current desirable endpoint of treatment against chronic hepatitis B virus infection (cHBV) is to achieve a functional cure, which is defined as HBsAg loss (sAg-L) with or without anti-HBs seroconversion. However, the immunological features that are associated with functional cure have not been studied in detail. METHODS: 172 cHBV patients (67 HBeAg+ and 105 HBeAg-), including 141 HBsAg retained (sAg-R) patients (115 chronic hepatitis and 26 asymptomatic carriers), 31 sAg-L patients, and 24 healthy individuals (vaccinated but not infected with HBV) were examined for their T cell phenotypic profile and HBV-specific T cell responses by flow cytometry. 18 cHBV patients with low serum HBsAg levels were also longitudinally followed for their T cell phenotypic profile and HBV-specific T cell responses up to 60 weeks. FINDINGS: sAg-L patients showed distinct CD4+ and CD8+ T cell phenotype fingerprints compared to those of sAg-R patients, as mainly indicated by the upregulation of HLA-DR on both CD4+ and CD8+ T cells, and a potent HBcAg-specific CD8+ T cell response. The changes in the T cell phenotype in cHBV patients were even more profound during rapid HBsAg decrease and was associated with interferon α treatment. The expression of HLA-DR (r = 0·3269, p = 0·0037), CD95 (r = 0·2796, p = 0·0151), CD40L (r = 0·2747, p = 0·0156), CTLA-4 (r = 0·2786, p = 0·0148), TIM-3 (r = 0·3082, p = 0·0068), CD107a (r = 0·3597, p = 0·0013) on CD4+ T cells, and HLA-DR (r = 0·3542, p = 0·0016), CD69 (r = 0·2507, p = 0·0279), CD107a (r = 0·2875, p = 0·0112) on CD8+ T cells were positively correlated with the rate of HBsAg decrease. The expression of HLA-DR (r = 0·2846, p = 0·0009) and CD95 (r = 0·2442, p = 0·0049) on CD8+ T cells were positively correlated with the magnitude of the HBcAg-specific T cell responses in cHBV patients. Importantly, CTLA-4, CD95 and CD107a expression on CD4+ T cells, as well as HLA-DR and TIM-3 expression on CD8+ T cells in combination with HBsAg quantification were identified as potential predictive factors for sAg-L within 48 weeks in cHBV patients. INTERPRETATION: The onset of HBsAg decrease and subsequent loss in cHBV patients on treatment is associated with significant alterations of both CD4+ and CD8+ T cell phenotypes. Characterization of the T cell phenotype in cHBV patients may present predicative value for sAg-L. FUNDING: National Natural Science Foundation of China, National Scientific and Technological Major Project of China, Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, "Double-First Class" Project for the International Cooperation Center on Infection and Immunity, HUST.


Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Hepatite B Crônica/sangue , Linfócitos T/imunologia , Adulto , Biomarcadores/sangue , Feminino , Citometria de Fluxo/métodos , Antígenos HLA-DR/imunologia , Hepatite B Crônica/imunologia , Humanos , Imunofenotipagem/métodos , Masculino
10.
Methods Mol Biol ; 2314: 261-271, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235657

RESUMO

Flow cytometry enables the measurement of tens of features on individual cells from complex mixtures. Flow cytometry enables high-throughput quantification of cell size, gene and protein expression. In the case of studies of host-pathogen interactions, this tool provides a facile way of identifying cells that have been successfully infected by a pathogen. Several recent technological advances have greatly improved throughput and the number of features that can be simultaneously monitored by this technique. Here, we describe common workflows to study Mycobacterium tuberculosis heterogeneity and host-M. tuberculosis interactions using flow cytometry and related technologies.


Assuntos
Citometria de Fluxo/métodos , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Macrófagos/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium tuberculosis/patogenicidade , Humanos , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/patologia
11.
Methods Mol Biol ; 2314: 385-398, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235664

RESUMO

The mycobacterial cell envelope includes a unique outer membrane, also known as the mycomembrane, which is the major defense barrier that confers intrinsic drug tolerance to Mycobacterium tuberculosis (Mtb) and related bacteria. The mycomembrane is typified by long-chain mycolic acids that are esterified to various acceptors, including: (1) trehalose, forming trehalose mono- and di-mycolate; (2) arabinogalactan, forming arabinogalactan-linked mycolates; and (3) in some species, protein serine residues, forming O-mycoloylated proteins. Synthetic trehalose and trehalose monomycolate analogs have been shown to specifically and metabolically incorporate into mycomembrane components, facilitating their analysis in native contexts and opening new avenues for the specific detection and therapeutic targeting of mycobacterial pathogens in complex settings. This chapter highlights trehalose-based probes that have been developed to date, briefly discusses their applications, and describes protocols for their use in mycobacteria research.


Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/análise , Trealose/química , Membrana Celular/química , Membrana Celular/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Imagem Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Ácidos Micólicos/metabolismo
12.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299367

RESUMO

The scope of application of carbon nanomaterials in biomedical, environmental and industrial fields is recently substantially increasing. Since in vitro toxicity testing is the first essential step for any commercial usage, it is crucial to have a reliable method to analyze the potentially harmful effects of carbon nanomaterials. Even though researchers already reported the interference of carbon nanomaterials with common toxicity assays, there is still, unfortunately, a large number of studies that neglect this fact. In this study, we investigated interference of four bio-promising carbon nanomaterials (graphene acid (GA), cyanographene (GCN), graphitic carbon nitride (g-C3N4) and carbon dots (QCDs)) in commonly used LIVE/DEAD assay. When a standard procedure was applied, materials caused various types of interference. While positively charged g-C3N4 and QCDs induced false results through the creation of free agglomerates and intrinsic fluorescence properties, negatively charged GA and GCN led to false signals due to the complex quenching effect of the fluorescent dye of a LIVE/DEAD kit. Thus, we developed a new approach using a specific gating strategy based on additional controls that successfully overcame all types of interference and lead to reliable results in LIVE/DEAD assay. We suggest that the newly developed procedure should be a mandatory tool for all in vitro flow cytometry assays of any class of carbon nanomaterials.


Assuntos
Carbono/toxicidade , Nanoestruturas/toxicidade , Células Cultivadas , Citometria de Fluxo/métodos , Fluorescência , Corantes Fluorescentes/toxicidade , Grafite/toxicidade , Humanos , Compostos de Nitrogênio/toxicidade , Pontos Quânticos/toxicidade
13.
Methods Mol Biol ; 2320: 35-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302646

RESUMO

The most common method for isolating cells of interest is an antibody method that recognizes cell surface antigens. However, specific surface antigens for many cell types have not been identified. We have developed the microRNA (miRNA)-responsive synthetic mRNA systems (miRNA switches), which isolate target cells based on endogenous miRNA activity. In this chapter, we describe protocols for isolating human pluripotent stem cell (hPSC)-derived cardiomyocytes using miRNA switches with or without cell sorting.


Assuntos
MicroRNAs/genética , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Separação Celular/métodos , Citometria de Fluxo/métodos , Humanos , RNA Mensageiro/genética
14.
Folia Histochem Cytobiol ; 59(2): 75-85, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097299

RESUMO

INTRODUCTION: Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs. MATERIAL AND METHODS: Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed. RESULTS: The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs. CONCLUSIONS: The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.


Assuntos
Antígenos CD/sangue , Antígenos HLA-DR/sangue , Linfócitos T Reguladores/química , Biomarcadores/sangue , Contagem de Linfócito CD4/métodos , Citometria de Fluxo/métodos , Fatores de Transcrição Forkhead/sangue , Doença Enxerto-Hospedeiro/sangue , Doença Enxerto-Hospedeiro/imunologia , Humanos , Linfócitos T Reguladores/classificação , Timo/imunologia
15.
Methods Mol Biol ; 2329: 195-204, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085224

RESUMO

CRISPR/Cas9 system is a powerful technique for genome editing and engineering but obtaining a sizeable population of edited cells can be challenging for some cell types. CRISPR/Cas9-induced cell cycle arrest is a possible cause of this barrier to efficient editing; thus, it is desirable to know the cell cycle progression profile of any given cell line or type of interest resulting from CRISPR/Cas9 treatment. Here we describe a flow cytometry-based assay that enables the determination of cell cycle progression in the presence of CRISPR/Cas9 treatment, in addition to the transfection and expression efficiencies of Cas9 vectors. This assay can also easily determine the effect of various interventions on obtaining a larger pool of Cas9-treated cells.


Assuntos
Ciclo Celular , Citometria de Fluxo/métodos , Edição de Genes/métodos , Sistemas CRISPR-Cas , Linhagem Celular , Química Click , Imunofluorescência , Humanos , Transfecção
16.
Methods Mol Biol ; 2323: 121-140, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086278

RESUMO

The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a profluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.


Assuntos
Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Citometria de Fluxo/métodos , Corantes Fluorescentes/análise , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , RNA de Transferência/genética , RNA/genética , Riboswitch/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compostos de Benzil , Clonagem Molecular/métodos , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imidazolinas , Isopropiltiogalactosídeo/farmacologia , Conformação de Ácido Nucleico , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Plasmídeos
17.
Eur J Immunol ; 51(8): 1992-2005, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34081326

RESUMO

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.


Assuntos
Mineração de Dados/métodos , Citometria de Fluxo/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , COVID-19/sangue , COVID-19/imunologia , Citocinas/metabolismo , Engenharia Genética , Humanos , Memória Imunológica , Imunofenotipagem , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , SARS-CoV-2/imunologia
18.
Methods Mol Biol ; 2326: 155-165, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097267

RESUMO

This chapter describes, in detail, the operational principles and experimental design to analyze the premature death of human red blood cells (RBCs; erythrocytes). Necrosis (i.e., hemolysis), eryptosis, and necroptosis are the three types of cell death thus far known to exist in RBCs, and distinctive markers of each are well established. Here, methods based on flow cytometry are presented in an easily reproducible form. Moreover, manipulation of incubation medium to promote or inhibit certain physiological phenomena, along with a step-by-step approach to examine membrane scrambling, cell volume, surface complexity, calcium activity, oxidative stress, and signal transduction pathways are also discussed.


Assuntos
Eriptose , Eritrócitos/citologia , Citometria de Fluxo/métodos , Hemólise , Necroptose , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Hemólise/efeitos dos fármacos , Humanos , Necroptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Testes de Toxicidade/métodos
19.
Methods Mol Biol ; 2276: 203-213, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060043

RESUMO

To evaluate how a cell responds to the external stimuli, treatment, or alteration of the microenvironment, the quantity and quality of mitochondria are commonly used as readouts. However, it is challenging to apply mitochondrial analysis to the samples that are composed of mixed cell populations originating from tissues or when multiple cell populations are of interest, using methods such as Western blot, electron microscopy, or extracellular flux analysis.Flow cytometry is a technique allowing the detection of individual cell status and its identity simultaneously when used in combination with surface markers. Here we describe how to combine mitochondria-specific dyes or the dyes targeting the superoxide produced by mitochondria with surface marker staining to measure the mitochondrial content and activity in live cells by flow cytometry. This method can be applied to all types of cells in suspension and is particularly useful for analysis of samples composed of heterogeneous cell populations.


Assuntos
Células Sanguíneas/metabolismo , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Baço/metabolismo , Superóxidos/metabolismo , Animais , Células Sanguíneas/citologia , Células Sanguíneas/ultraestrutura , Humanos , Baço/citologia , Baço/ultraestrutura
20.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072360

RESUMO

For many years, oncological clinical trials have taken advantage of dendritic cells (DC) for the design of DC-based cellular therapies. This has required the design of suitable quality control assays to evaluate the potency of these products. The purpose of our work was to develop and validate a novel bioassay that uses flow cytometry as a read-out measurement. In this method, CD3+ cells are labeled with a fluorescent dye and the DC costimulatory activity is measured by the degree of T cell proliferation caused by the DC-T cell interaction. The validation of the method was achieved by the evaluation of essential analytical parameters defined by international guidelines. Our results demonstrated that the method could be considered specific, selective, and robust. The comparison between measured values and estimated true values confirmed a high level of accuracy and a lack of systematic error. Repeated experiments have shown the reproducibility of the assay and the proportionality between the potency and the DC amount has proven its linearity. Our results suggest that the method is compliant with the guidelines and could be adopted as a quality control assay or batch-release testing within GMP facilities.


Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Biomarcadores , Vacinas Anticâncer/uso terapêutico , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Linfócitos T/imunologia , Linfócitos T/metabolismo
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