Work in the Barton lab was supported by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT RP110471). This research was supported in part, by a training grant fellowship for Ryan L. McCarthy from the National Institutes of Health Training Program in Molecular Genetics 5 T32 CA009299. The core facility that maintains and runs the mass cytometry machine is supported by CPRIT grant (RP121010) and NIH core grant (CA016672).

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The authors have nothing to disclose.

O protocolo aqui apresentado tem sido empregue com sucesso para o tratamento de várias linhas de cultura de células (H1 e H9 hESCs, mESCs, MCF7, HEK 293, KBM5, HMEC, MCF10A) e amostras de tecido primárias (de medula óssea de ratinho, rato fígado embrionário, rato adulto fígado, tumor de rato). Independentemente da fonte, qualquer tecido que pode ser dissociado em células individuais, preservando o estado celular devem ser passíveis de análise por citometria de massa, mas pode requerer alguma optimização prot...

Citometria permite a medição simultânea de múltiplos alvos de anticorpos a um nel de cula ica em grandes populações de células. Em citometria de fluxo baseado em fluorescência tradicional, o número de parâmetros que podem ser quantificados é limitada pela sobreposição espectral entre o espectro de emissão de vários fluoróforos, que requer cálculos de compensação cada vez mais complexos, como o número de parâmetros aumenta. Estas limitações são abordados por massa citometria, onde anticorpos de metal-conjugado pesados ​​são detectadas e quantificadas por tempo de voo (TOF) espectrometria de massa para expandir o número de parâmetros recolhidos ao mesmo tempo e produzir um perfil de proteína de abundância alta dimensional para cada célula individual. Uma compreensão básica do funcionamento da massa citometria instrumento é benéfico para o usuário e pode ser essencial para solução de problemas. Para uma descrição mais completa do ajuste e executando uma máquina de citometria de massa,ver o manuscrito relacionados 1. Resumidamente, uma amostra celular é marcada com um painel de anticorpos conjugados de metal de segmentação marcadores da superfície celular, proteínas citoplasmáticas, proteínas nucleares, proteínas ligadas a cromatina ou outros epitopos de interesse (Figura 1-I). As células marcadas são carregados na máquina, quer uma de cada vez através de injecção manual ou usando um amostrador automático que amostras a partir de uma placa de 96 poços. As células carregadas foram injectados através de um nebulizador, a qual gera uma pulverização de gotículas de líquido que encapsulam as células (Figura 1ii). Esta pulverização é posicionado de modo que as células são ionizados por uma tocha de plasma de árgon. Este ionização gera uma nuvem de partículas formada por todos os átomos constituintes de cada uma das células (Figura 1iii). Como esta nuvem de partículas viaja para o detector, átomos de baixa massa atómica são separados dos iões de massa elevadas por um filtro de massa de quadrupolo. Os restantes iões de massa de alta continuar para o detector, onde o abunddade de cada isótopo é quantificado (Figura 1iv). Os dados brutos recolhidos pelo detector, são analisados ​​pelo software do instrumento de citometria de massa para identificar eventos celulares. Para cada evento celular identificado, o sinal detectado em cada canal é quantificado e guardada num ficheiro de saída um .fcs. Os canais de massa utilizados para detectar os anticorpos de metal pesado conjugados apresentam um mínimo de sobreposição espectral, que varia de 0-4%, com a maioria das contribuições abaixo de 1%. Devido a essa baixa diafonia entre canais, geralmente não é necessário para transformar os dados com uma matriz de compensação antes da análise 2. No entanto, deve ser tomado cuidado durante a concepção do painel experimental de anticorpos para assegurar que um anticorpo com um epitopo de alta abundância não é atribuído a um canal de massa que contribui sinal a um canal atribuído a um anticorpo de baixa abundância, pois isso pode criar uma população artificialmente elevados no canal de receber a contribuição de sinal. <p clburro = &quot;jove_content&quot;&gt; A preparação exacta de amostras para análise de citometria de massa é altamente dependente dos tipos de amostras a ser recolhidos e a hipótese experimental a ser testado. Nós fornecem exemplos de dois protocolos para a colheita de diferentes tipos de células (células estaminais embrionárias humanas) e células primárias (de ratinho de tumor da mama do isolado de células epiteliais). Ao começar um estudo de citometria de massa, é aconselhável primeiro fazer uma pequena experiência piloto para garantir que todos os protocolos e anticorpos a serem utilizados estão a trabalhar bem nas mãos do investigador. Isto é especialmente essencial quando estão a ser utilizados anticorpos costume conjugados, como a reacção de redução parcial durante a conjugação anticorpo tem o potencial de alterar a afinidade do anticorpo; portanto, cada anticorpo costume precisa ser validado empiricamente para garantir a precisão dos dados. Outras considerações incluem a determinação de se os anticorpos da superfície celular para ser usado no ensaio irá reconhecer os seus epitopos após fixação ou, se need para ser aplicado às células ao vivo. Alguns fixação pode evitar coloração adequada com anticorpos de superfície celular como é conhecida para ocorrer com o péptido-MHC de coloração 3, 4. Realizando a coloração de anticorpos de superfície celular em células vivas pode ser necessária para alguns anticorpos, mas este opõe-se a opção de código de barras de antes da marcação do anticorpo como a maioria dos métodos de códigos de barras são realizadas após a fixação 5, embora o código de barras à base de anticorpo 6, 7 é uma excepção. Ao determinar o número de células a ser preparados para análise, é importante saber que apenas uma fracção das células carregadas dentro da máquina será detectado e produzir dados. Esta perda é principalmente devido a ineficiências quando o nebulizador pulveriza a amostra a tocha. A perda exata cento depende da massa citometria de configuração da máquina e tipo de célula, mas pode variar de 50-85% e deve serconsiderado na concepção do experimento. Este protocolo foi optimizado para 2x10 6 culas por amostra mas pode ser adaptado para o processamento de tamanhos de amostras menores ou maiores. Este protocolo pode ser utilizado com modificações mínimas para tamanhos de amostra a partir de 1 x 10 6 4 x 10 6, no entanto, é crítico que o tamanho da amostra ser mantidos consistentes dentro de uma experiência. As alterações no número de células pode afectar a intensidade de código de barras e anticorpo coloração e deve ser mantido aproximadamente constante entre as amostras a serem comparadas directamente. Alguns procedimentos, tais como a rotulagem de células mortas e de marcao de ADN, deve ser levada a cabo na grande maioria, se não todos os modelos experimentais. A marcação das células mortas em experiências de análise de marcadores de superfície apenas pode ser conseguida utilizando Rh103, mas Rh103 deve ser evitado para experiências onde permeabilização é necessária, uma vez que resulta em perda de coloração. Para experiências envolvendo permeabilização, é aconselhável utilizar a cisplatina <s-se classe = &quot;refex&quot;&gt; 8 e, quando possível, um anticorpo que reconhece a PARP clivada ou caspase para detectar células apoptóticas e mortas. Amostras de código de barras pode reduzir o consumo de anticorpo, diminuir o tempo de aquisição e eliminar amostra-a-amostra variabilidade 9. O processo de barcoding envolve a aplicação de um código específico de amostra única para todas as células, que é usado para atribuir cada célula para sua amostra de origem. Depois de código de barras das amostras podem ser reunidas antes da coloração do anticorpo, um processo que assegura que todas as amostras são igualmente coradas. Para minimizar o erro experimental, é prudente de código de barras e reunir as amostras que estão a ser comparados directamente uns com os outros. Actualmente existem vários métodos para o código de barras de amostras para análise de citometria de massa 5, 6, 7, dois dos quais são aqui apresentados (ver abaixo). Com Reagen atualmente disponívelts é possível quantificar a abundância de 38 alvos de anticorpos, identificar as células na fase S 10, distinguir as células vivas e mortas 8 e medir os níveis celulares de hipoxia 11 numa experiência multiplexado de múltiplas amostras. Esta capacidade de recolher os dados de uma única célula elevado de parâmetros para as populações de células grandes permite uma melhor perfis de populações de células altamente heterogéneos e já produziu um número de novos conhecimentos biológicos 12, 13, 14, 15, 16. Destilando os dados complexo resultante à informação interpretável tem exigido o uso de algoritmos computacionais incluindo Spanning Tree Progressão da densidade normalizados Eventos (PÁ) 17 e vişne 18. Este artigo apresenta uma acessíveisvisão geral de como projetar e realizar uma massa citometria de experiência e introduz análise básica de massa citometria de dados.

<table><tbody><tr><td>mTeSR1 medium kit</td><td>Stem Cell technologies</td><td>05850</td><td>Warm at room temperature before use</td></tr><tr><td>DMEM F-12</td><td>ThermoFisher</td><td>11330-032</td><td>Warm at 37&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Accutase</td><td>Stem Cell technologies</td><td>07920</td><td>Warm at 37&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Bovine serum albumin</td><td>Equitech</td><td>BAH62</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Cell ID Pt194</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cell ID Pt195</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cell ID Pt196</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cisplatin</td><td>Enzo Life Sciences</td><td>ALX-400-040-M250</td><td>Soluble to 25&amp;nbsp;mg/mL in DMSO</td></tr><tr><td>Cell-ID 20-plex Pd Barcoding Kit</td><td>Fluidigm</td><td>201060</td><td></td></tr><tr><td>5-Iodo-2&#039;-deoxyuridine</td><td>Sigma-Aldrich</td><td>I7125</td><td>Soluble to 74&amp;nbsp;mg/mL in 0.2&amp;nbsp;N NaOH</td></tr><tr><td>Rhodium 103 intercelating agent</td><td>Fluidigm</td><td>201103A</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>BP1105-4</td><td>Chill at -20&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>5 mL round bottom tubes</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>5 mL 35 &amp;#956;m filter cap tubes</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>EQ four element calibration beads</td><td>Fluidigm</td><td>201078</td><td></td></tr><tr><td>Hyaluronidase Type I-S</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C9891</td><td></td></tr><tr><td>Disposable Scalpel #10</td><td>Sigma-Aldrich</td><td>Z69239</td><td></td></tr></tbody></table>

sample preparation for mass cytometry analysis

Mass citometria utiliza anticorpos conjugados com etiquetas de metal pesado, uma abordagem que aumentou muito o número de parâmetros e oportunidades para a análise profunda bem além do que é possível com fluxo baseado em fluorescência convencional citometria. Tal como acontece com qualquer nova tecnologia, há passos críticos que ajudam a garantir a geração confiável de dados de alta qualidade. Apresentada aqui é um protocolo optimizado que incorpora várias técnicas para o processamento de amostras de células para análise de citometria de massa. Os métodos descritos aqui vai ajudar o utilizador a evitar erros comuns e alcançar resultados consistentes, minimizando variabilidade, que pode levar a dados incorrectos. Para informar desenho experimental, a lógica subjacente a passos opcionais ou alternativos no protocolo e a sua eficácia na descoberta de novas descobertas na biologia do sistema a ser investigada é coberto. Finalmente, os dados representativo é apresentado para ilustrar os resultados esperados a partir da Presente técnicasd aqui.

O protocolo aqui apresentado pode ser amplamente aplicada a uma vasta variedade de amostras de células cultivadas e primárias, com apenas pequenas modificações. Dependendo dos requisitos de uma experiência, que pode ser realizada de uma maneira modular, quando certos elementos, tais como o código de barras ou de coloração da superfície celular, não são necessários. Utilizado na sua totalidade, este protocolo permite a preparação de massa multiplexado por citometria de amost...

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Sample Preparation for Mass Cytometry Analysis

This article describes the collection and processing of samples for mass cytometry analysis.

Preparação de Amostras para Análise Mass Citometria

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

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Biology

16.0K visualizações.  The University of Texas MD Anderson Cancer Center. This article describes the collection and processing of samples for mass cytometry analysis.

Vídeo: Sample Preparation for Mass Cytometry Analysis

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Citometria de massa: Protocolo para Ajuste Diário e amostras de células em execução em um citômetro de Massa CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Bioengenharia

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

Isolação e mancha de keratinocytes da pele do rato para a análise específica do ciclo de pilha da expressão celular da proteína pela citometria maciça

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Biologia do Desenvolvimento

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Coleção, isolamento e Citometria de Fluxo de Análise de Amostras endocervix Humano

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Medicina

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Identificação e Isolamento de Progenitores Eritróides da Unidade Formadora de Explosão e da Unidade Formadora de Colônias de Tecido de Camundongo por Citometria de Fluxo

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Análise de célula única do imunofenótipo e produção de citocinas no sangue periférico através de citometria de massa

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Imunologia e Infecção

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparação de medula óssea inteira para análise de citometria de massa de células de linhagem de neutrófilos

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Estudos metabólicos de baixa entrada em tipos de células de camundongos

Targeted Metabolomics on Rare Primary Cells

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Autor em destaque: Avançando na análise metabolômica de células-tronco hematopoiéticas raras 

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

Metabolômica direcionada em células primárias raras

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

Preparação de Amostras para Análise Mass Citometria

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