1. A Importância da contagem de células <ol><li> As células de contagem é um passo necessário, mas tedioso para in vitro de cultura de células. </li><li> Concentrações de células consistente garantir a reprodutibilidade e precisão experimental. </li><li> Contagem de células são importantes para a vigilância da saúde e taxa de proliferação celular, avaliando imortalização ou transformação, para posterior semeadura células experimentos de transfecção, ou infecção, e preparação para celular baseado em ensaios. </li><li> Apesar da necessidade de velocidade e precisão na contagem de células, 71% de 400 pesquisadores entrevistaram contagem de células utilizando um hemocitômetro. Enquanto hemocytometry é barato, é trabalhoso e sujeito a viés de usuário e uso indevido, o que resulta na contagem imprecisa. </li><li> Fontes de erro em hemocytometry incluem: distribuição desigual de células na amostra, muitas ou poucas células na amostra, decisões subjetivas quanto a saber se uma determinada célula cai dentro da área de contagem definido, a contaminação do hemocitômetro, de usuário para usuário variação , variação e da taxa de hemocitômetro de enchimento. </li><li> Para aliviar o tédio associado com a contagem manual, 29% dos pesquisadores contagem de células usando dispositivos de células automatizado de contagem. No entanto, para a maioria dos pesquisadores, o preço associado a essas grandes instrumentos de bancada é uma barreira à sua utilização. </li><li> O contador de células Scepter é um dispositivo automático de mão que oferece a automação e precisão de contagem Coulter, a um custo relativamente baixo. </li><li> O sistema emprega o princípio Coulter de impedância baseado em detecção de partículas em um formato miniaturizado usando uma combinação de hardware analógico e digital para a detecção, processamento de sinais, dados, armazenamento e visualização gráfica. </li><li> O sensor disponível é projetado para receber uma microfabricated, zona de células-sensing que possibilita a discriminação por tamanho de célula e do volume celular em sub-micron e sub-picoliter resolução. </li><li> Reforçada com precisão líquido de manipulação de canais e eletrônica, o contador de células Scepter graficamente exibe estatísticas de população de células como um histograma. </li></ol> 2. Preparação de uma suspensão de células isoladas de Contagem <ol><li> Para usar o contador de células Scepter, comece diluindo uma suspensão única célula de células, em tampão fosfato salino. A concentração de células por mL 10,000-500,000 é o ideal. </li><li> Transferir 100 mL de células diluída para um microtubo de 1,5 mL. </li></ol> 3. Realizar a contagem de células usando o Scepter <ol><li> Ligar o contador de células Scepter pressionando e segurando a alternância na parte de trás do instrumento e aguarde as instruções na tela para aparecer. </li><li> Quando solicitado, conecte um sensor para o fim da unidade Scepter com o painel de eletrodo sensor de virada para a frente do instrumento. </li><li> Sensores cetro consistem em uma câmara de amostragem precisamente modulada, com uma zona de sensoriamento eletrônico e integrado de células-sensing eletrodos. Os sensores permitem discriminar tamanho da célula em sub-micron, resolução e volume de células em sub-picoliter resolução. </li><li> Sensor de engenharia consistente, garante que os volumes de amostra igual são contados e as medições são feitas tamanho exato. </li><li> Uma vez que o sensor é conectado, executar cada passo do processo de contagem de acordo com as instruções na tela da seguinte forma: </li><li> Quando a tela lê &quot;Mantenha o êmbolo para começar&quot;, o êmbolo. A tela, então, ler &quot;submergir o sensor.&quot; Submergir o sensor na solução de suspensão de células e solte o êmbolo para aspirar a suspensão celular para o sensor. </li><li> O êmbolo age como um sinal elétrico e vai chamar a amostra mais o volume morto no sensor. A velocidade ou precisão de mergulhar não importa como faz com pipetagem. Este processo vai demorar vários segundos. Quando estiver pronto, o instrumento emite um sinal sonoro, e &quot;Amostra carregado&quot; será exibido na tela. </li><li> Uma vez que a amostra é carregado no sensor, ela é puxada através da zona de detecção para avaliar o tamanho das células e volume da célula e, em seguida pelos microcanais. Uma vez que 50 mL de amostra é contado o sensor diz ao instrumento para parar a contagem. </li><li> Após a contagem estiver concluída, a tela do instrumento irá ler &quot;contagem completa. Por favor, remova o sensor e descarte.&quot; Siga o prompt, e remover e descartar o sensor. </li><li> O instrumento irá exibir um histograma de tamanho de célula ou de diâmetro em sua tela, bem como a concentração de células por ml. </li></ol> 4. Análise de Dados <ol><li> Após a contagem é completo eo histograma é mostrada no aparelho, aplicar portas pressionando o botão de alternância. Selecione &quot;gating auto&quot; ou &quot;último uso&quot; quer definir automaticamente o portão com base no perfil do histograma, ou para usar as portas a partir da contagem anterior. </li><li> Independentemente da seleção, os portões podem ser movidos manualmente para ajustar os dados. Pressione o botão alternar novamente para selecionar a porta a esquerda e para rolar aggle para mover o portão, como desejado. </li><li> Em seguida, pressione a alternar novamente para ativar a porta à direita e percorrer a alternar para colocar o portão certo. Clique novamente, uma nova célula de concentração, o volume celular médio e diâmetro será exibida. </li><li> A alternância também pode ser movido para qualquer lado para alternar a exibição entre data, hora, a média do tamanho das células, ea média do volume da célula. </li><li> Até 72 histogramas podem ser armazenados no próprio instrumento. Para carregar os dados para um computador pessoal, anexe o instrumento através da porta USB do cabo na tela. </li><li> No PC, clique duas vezes sobre a Aplicação de Software Scepter para o lançamento do software, em seguida, clique em &quot;connect&quot;. Todas as acusações serão automaticamente porta para o software. </li><li> O Software Aplicativo Scepter mostra o histograma detalhando a distribuição da população da cultura em volume ou diâmetro. Arquivos baixados poderão ser exportados para o Microsoft Excel, onde os dados brutos serão exibidos em caixas de diâmetros célula especificada ou volumes de células. </li></ol> 5. Resultados representante <ol><li> Dezenove diferentes linhagens celulares foram preparados para contagem e concentrações de partida teóricos foram determinados utilizando um contador Coulter. Estas suspensões de células foram então diluída para obter suspensões de células em concentrações célula teórica. </li><li> O contador de células Scepter foi então usada para determinar as concentrações de células. A Figura 1 mostra cinco diluições que foram medidos para cinco linhagens de células representante junto com a concentração de células teórica. </li><li> O alto grau de linearidade, conforme indicado pelos valores R2, demonstra que a contagem Scepter é um método confiável para as linhagens celulares testadas, através de uma ampla faixa operacional linear. </li><li> A precisão global relativa de contagem Scepter é demonstrado na Figura 2, em que os dados obtidos da contagem Scepter são comparados com as contagens obtidas Contando Coulter. </li><li> O log da contagem de células Scepter é plotado contra o log da contagem de Coulter, e mostra que, em todas as linhagens, conta Scepter coincidir com contagem de Coulter. </li><li> Contagem de 19 linhas de células foram realizadas usando um Z2 Coulter Counter, o contador de células Scepter, um contador automático baseado em visão, como Vi-Cell ou sistema de incontáveis, e um hemocytemeter. </li><li> As contagens foram realizadas de acordo com as instruções do fabricante, usando a mesma célula de partida de suspensão e diluição idênticos. Os resultados mostrados na Figura 3 demonstram que as concentrações de células medido pela contagem Scepter coincidem com concentrações de células teórica com alta linearidade. </li><li> O percentual de variação, ou CV, foi calculado para cada diluição de cada linha de células para cada sistema de contagem. A Figura 4 mostra os currículos% em média mais de 19 linhagens de células com respeito à concentração de células e método de contagem. </li><li> Dentro da faixa de operação, o contador de células Scepter é mais preciso do que a visão baseada contagem hemocitômetro, exibindo menores desvios-padrão e menores coeficientes médios de variação. </li><li> Contagem cetro é dez vezes mais rápido que hemocytometry e mais rápido que outros contadores automáticos também. Na Figura 5, Sf9, MCF7 e HEK293 células foram contadas utilizando os métodos descritos neste vídeo. </li><li> O tempo médio necessário para medir a concentração de células foi registrada. Note-se que hemocytometry foi realizada apenas uma vez por amostra. </li><li> Cetro faz medições com base no tamanho da célula. Assim, quaisquer alterações na morfologia celular podem ser capturados no histograma. Culturas saudáveis ​​exibir um belo sino distribuição de Gauss em forma, enquanto as células saudáveis, lyse, e causar uma mudança no histograma para a esquerda (Figura 6, painel esquerdo). </li><li> Da mesma forma, quando uma população de 50% de células mortas foram misturados com 50% de células vivas, as mudanças histograma também foram observados (Figura 6, painel direito). </li></ol><img src="/files/ftp_upload/2204/2204fig1.jpg" alt="Figura 1" /><img src="/files/ftp_upload/2204/2204fig2.jpg" alt="Figura 2" /><img src="/files/ftp_upload/2204/2204fig3.jpg" alt="Figura 3" /><img src="/files/ftp_upload/2204/2204fig4.jpg" alt="Figura 4" /><img src="/files/ftp_upload/2204/2204fig5.jpg" alt="Figura 5" /><img src="/files/ftp_upload/2204/2204fig6.jpg" alt="Figura 6" /><img src="/files/ftp_upload/2204/2204fig7.jpg" alt="Figura 7" />

<ol>
	<li>Barghshoon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Counting Survey. , (2009).</li><li>Tucker, K. G., Chalder, S., al-Rubeai, M., Thomas, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+hybridoma+cell+number,+viability,+and+morphology+using+fully+automated+image+analysis.">Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.</a> Enzyme Microb Technol. 16 (1), 29-35 (1994).</li><li>Houwen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fifty years of hematology innovation: the Coulter principle. , (2003).</li></ol>

Kathleen Ongena, Chandreyee Das, Janet L. Smith, Sónia Gil, e Grace Johnston são empregados por Millipore, que produz os instrumentos utilizados neste artigo.

Comparando o desempenho do contador de células Scepter com resultados de outros métodos de contagem, podemos concluir que este novo handheld, contador de células automatizado oferece contagens de células precisa, rápida e confiável através de uma ampla faixa operacional. A funcionalidade de contagem Scepter é um resultado da tecnologia de engenharia de precisão embutido na ponta do sensor ea instrumentação sofisticada contagem baseada no princípio de Coulter. A qualidade de desempenho sugere Scepter contando como método para aumentar a velocidade e melhorar a reprodutibilidade de ensaios baseados em células. O formato handheld permite tédio rápida direita livre contagem na capa de cultura de células.

<table border="1"><tbody><tr><td> Nome do material </td><td> Companhia </td><td> Número Catálogo </td><td> Comentário </td></tr><tr><td> Cetro 2,0 contador de células automatizado Handheld com 40 sensores Scepter mM </td><td> EMD Millipore </td><td> PHCC20040 </td><td></td></tr><tr><td> Cetro 2,0 contador de células automatizado Handheld com 60 sensores Scepter mM </td><td> EMD Millipore </td><td> PHCC20060 </td><td></td></tr><tr><td> Sensores cetro, 60 mm </td><td> EMD Millipore </td><td> PHCC60050 </td><td> Pacote de 50 de sensores </td></tr><tr><td> Sensores cetro, 40 mM </td><td> EMD Millipore </td><td> PHCC40050 </td><td> Pacote de 50 de sensores </td></tr><tr><td> Universal Power Adapter </td><td> EMD Millipore </td><td> PHCCPOWER </td><td> </td></tr><tr><td> Cetro de O-Ring Kit </td><td> EMD Millipore </td><td> PHCCOCLIP </td><td> 2 O-rings e uma tampa do filtro </td></tr></tbody></table>

determining cell number during cell culture using the scepter cell counter

Contagem de células é muitas vezes um passo necessário, mas tedioso para in vitro de cultura de células. Concentrações de células consistente garantir a reprodutibilidade e precisão experimental. Contagem de células são importantes para a vigilância da saúde e taxa de proliferação celular, avaliando imortalização ou transformação, para posterior semeadura células experimentos de transfecção, ou infecção, e preparação para celular baseado em ensaios. É importante que as contagens de células ser precisa, consistente e rápido, especialmente para medições quantitativas de respostas celulares. Apesar desta necessidade de rapidez e precisão na contagem de células, 71% de 400 pesquisadores entrevistaram uma contagem de células que usando um hemocitômetro. Enquanto hemocytometry é barato, é trabalhoso e sujeito a viés de usuário e uso indevido, o que resulta na contagem imprecisa. Hemocytometers são feitas de vidro óptico especial em que suspensões de células são carregados em volumes especificados e contadas sob um microscópio. Fontes de erros na hemocytometry incluem: distribuição desigual de células na amostra, muitas ou poucas células na amostra, decisões subjetivas quanto a saber se uma determinada célula cai dentro da área de contagem definido, a contaminação do hemocitômetro, de usuário para usuário variação e variação da taxa de enchimento hemocitômetro 2. Para aliviar o tédio associado com a contagem manual, 29% dos pesquisadores contagem de células usando células automatizado de contagem dispositivos, que incluem a visão baseada em contadores, sistemas que detectam as células usando o princípio Coulter, ou citometria de fluxo 1. Para a maioria dos pesquisadores, a principal barreira ao uso de um sistema automatizado é o preço associado a essas grandes instrumentos de bancada 1. O contador de células Scepter é um dispositivo automático de mão que oferece a automação e precisão de contagem Coulter, a um custo relativamente baixo. O sistema emprega o princípio Coulter de impedância baseado em detecção de partículas 3 em um formato miniaturizado usando uma combinação de hardware analógico e digital para a detecção, processamento de sinais, armazenamento de dados e visualização gráfica. A ponteira descartável é projetada com um microfabricated, zona de células-sensing que possibilita a discriminação por tamanho de célula e do volume celular em sub-micron e sub-picoliter resolução. Reforçada com precisão líquido de manipulação de canais e eletrônica, o contador de células Scepter relatórios de estatísticas de células população graficamente como um histograma.

Watch this Scientific Journal Video about Determining Cell Number During Cell Culture using the Scepter Cell Counter at JoVE.com

Determining Cell Number During Cell Culture using the Scepter Cell Counter

O contador de células Scepter é um dispositivo portátil automático que pode ser usado para contagem de células, monitor de diâmetro celular e volume, e ser usado para verificar a saúde e qualidade de populações celulares de uma cultura para outra.

Determinação do Número celular durante Cultura Celular usando o contador de células Scepter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

determining-cell-number-during-cell-culture-using-scepter-cell

Research

JoVE Journal

Biology

37.8K visualizações.  Millipore Inc. O contador de células Scepter é um dispositivo portátil automático que pode ser usado para contagem de células, monitor de diâmetro celular e volume, e ser usado para verificar a saúde e qualidade de populações celulares de uma cultura para outra.

Vídeo: Determining Cell Number During Cell Culture using the Scepter Cell Counter

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Células ES: Cultura A partir de células congeladas

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Biologia

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Mudanças Dinâmicas de monitoramento dos níveis de cálcio mitocondrial durante a apoptose através de um sensor de cálcio geneticamente codificado

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirus mediada Durante Knockdown Ex Vivo Eritropoese de Células-Tronco Hematopoéticas

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Determinação da contagem de células de mamíferos, tamanho de célula e Saúde célula usando o Moxi Z Mini contador de células automatizado

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Rastreamento de celular utilizando proteínas Photoconvertible Durante o Desenvolvimento Zebrafish

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Explantes blastômero para testar Compromisso destino celular durante o desenvolvimento embrionário

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Comparação de três métodos diferentes de determinação da proliferação celular em linhas celulares de cancro da mama

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Viver a microscopia de fluorescência da célula para observar os processos essenciais durante o crescimento de células microbianas

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.