1. La importancia de contar con celular <ol><li> Contando las células es un paso necesario pero aburrido para el cultivo celular in vitro. </li><li> Concentraciones consistentes celular asegurar la reproducibilidad experimental y precisión. </li><li> Los recuentos de células son importantes para la vigilancia de la salud celular y la tasa de proliferación, la evaluación de la inmortalización y transformación, la siembra de células de experimentos posteriores, transfección o infección, y la preparación de ensayos basados ​​en células. </li><li> A pesar de la necesidad de velocidad y la precisión en el recuento de células, el 71% de los investigadores encuestaron a 400 células cuentan con un hemocitómetro. Mientras hemocytometry es barato, es laborioso y sujeto a un sesgo de usuario y el uso indebido, que se traduce en cuenta inexacta. </li><li> Fuentes de error en hemocytometry incluyen: la distribución desigual de células en la muestra, de más o de muy pocas células en la muestra, las decisiones subjetivas en cuanto a si una célula dada entra en el área de conteo definido, la contaminación del hemocitómetro, de usuario a usuario variación , y la variación de la tasa de llenado hemocitómetro. </li><li> Para aliviar el tedio asociado con el conteo manual, el 29% de los investigadores cuentan las células se utilizan dispositivos automáticos conteo de células. Sin embargo, para la mayoría de los investigadores, el precio asociado con estos grandes de banco de instrumentos es una barrera para su uso. </li><li> El contador de células cetro es un dispositivo automático de mano que ofrece la automatización y la exactitud del conteo Coulter, a un costo relativamente bajo. </li><li> El sistema utiliza el principio de la detección de partículas Coulter impedancia basada en un formato de miniatura utilizando una combinación de hardware analógico y digital para la detección, procesamiento de señales, almacenamiento de datos y visualización gráfica. </li><li> El sensor desechable se ha diseñado con un microfabricated, la célula de detección de la zona que permite la discriminación por tamaño de la celda y el volumen celular en el sub-micrones y la resolución de sub-picolitros. </li><li> Mejorado con una precisión de manejo de líquidos de los canales y la electrónica, el contador de células cetro muestra gráficamente las estadísticas de la población de células en forma de histograma. </li></ol> 2. Preparación de una suspensión de una sola célula para el recuento <ol><li> Para utilizar el contador celular Scepter, empezar por la dilución de una suspensión de células individuales de las células, en tampón fosfato salino. Una concentración de 10,000-500,000 células por ml es lo óptimo. </li><li> Transferencia de 100 L de células diluidas a un tubo de microcentrífuga de 1,5 ml. </li></ol> 3. Realizar recuentos de células usando el cetro <ol><li> Encienda el contador de células Cetro pulsando y manteniendo pulsado el conmutador en la parte posterior del instrumento y esperar las instrucciones en pantalla para aparecer. </li><li> Cuando se le solicite, conecte un sensor al final de la unidad cetro con el panel de electrodo sensor mirando hacia la parte frontal del instrumento. </li><li> Sensores cetro consiste en una cámara de precisión del muestreo, moldeado, con una zona de detección electrónica e integrada electrodos de detección de células. Los sensores permiten discriminar el tamaño de celda en el sub-micras de resolución, y el volumen celular en el sub-picolitros resolución. </li><li> Ingeniería sensor consistente, asegura que volúmenes iguales de la muestra se cuentan y las medidas exactas de tamaño se hacen. </li><li> Una vez que el sensor está conectado, lleve a cabo cada paso del proceso de conteo de acuerdo a las instrucciones de la pantalla de la siguiente manera: </li><li> Cuando la pantalla se lee &quot;Mantenga el émbolo para empezar&quot;, el émbolo. La pantalla mostrará &quot;sumergir el sensor.&quot; Sumerja el sensor en la solución de suspensión celular y la liberación del émbolo para aspirar la suspensión de células en el sensor. </li><li> El émbolo actúa como una señal eléctrica y extraer la muestra, más volumen muerto en el sensor. La velocidad y precisión de sumergirse, no importa como lo hace con la pipeta. Este proceso tardará varios segundos. Cuando esté listo, el instrumento emitirá un pitido, y &quot;Ejemplo de carga&quot; se mostrará en la pantalla. </li><li> Una vez que la muestra se carga en el sensor que se dibuja a través de la zona de detección para evaluar el tamaño celular y el volumen celular, luego a través de los microcanales. Una vez que 50 L de la muestra se cuenta el sensor indica el instrumento para detener el conteo. </li><li> Tras el recuento se ha completado, la pantalla del instrumento mostrará &quot;Conteo Completo. Retire y deseche el sensor&quot;. Siga las indicaciones, y retirar y desechar el sensor. </li><li> El instrumento mostrará un histograma de tamaño de la celda o el diámetro de su pantalla, así como la concentración de células por ml. </li></ol> 4. Análisis de Datos <ol><li> Tras el recuento se ha completado y el histograma se muestra en el instrumento, se aplican las puertas pulsando el botón de activación. Seleccione &quot;gating auto&quot; o &quot;último uso&quot; a cualquiera ajusta automáticamente la puerta en función del perfil de histograma, o para usar las puertas de la cuenta anterior. </li><li> Independientemente de la selección, las puertas se pueden mover manualmente para ajustar los datos. Presione el botón de alternar de nuevo para seleccionar la puerta de la izquierda y vaya a laggle para mover la puerta a su gusto. </li><li> A continuación, pulse la palanca de nuevo para activar la puerta de la derecha y desplazar la palanca para colocar la puerta de la derecha. Haga clic de nuevo, una nueva concentración de células, el volumen celular y el diámetro en la pantalla. </li><li> La palanca también se puede mover hacia los lados para cambiar la visualización entre la fecha, la hora, la media de tamaño de la celda, y el volumen celular. </li><li> Hasta 72 histogramas se pueden almacenar en el propio instrumento. Para cargar los datos a un ordenador personal, conecte el instrumento a través del puerto USB del cable en la pantalla. </li><li> En el PC, haga doble clic sobre la Aplicación de Software Cetro para iniciar el software y haga clic en &quot;conectar&quot;. Todos los cargos de forma automática el puerto con el software. </li><li> El software de aplicación cetro muestra el histograma que detalla la distribución de la población de la cultura, ya sea en volumen o diámetro. Los archivos descargados pueden ser exportados a Microsoft Excel, donde los datos en bruto se mostrará en los contenedores de los diámetros de celda especificada o volúmenes de la célula. </li></ol> 5. Resultados representante <ol><li> Diecinueve diferentes líneas celulares se prepararon para el recuento y la concentración de partida teórico se determinaron mediante un contador Coulter. Estas suspensiones celulares fueron luego más diluidos para obtener suspensiones celulares en las concentraciones de células teórico. </li><li> El contador de células cetro se utilizó para determinar las concentraciones de células. La figura 1 muestra cinco diluciones que se midieron durante 5 líneas celulares representativas, junto con la concentración de células teórico. </li><li> El alto grado de linealidad, como se indica en los valores R2, demuestra que el conteo de Cetro es un método confiable para las líneas celulares estudiadas, a través de un amplio rango de funcionamiento lineal. </li><li> La precisión global relativa de contar cetro se demuestra en la figura 2, en el que los datos obtenidos de contar cetro se comparan con los recuentos obtenidos a partir de contar Coulter. </li><li> El registro de los recuentos de células cetro se traza contra el registro de la cuenta Coulter, y muestra que, en todas las líneas celulares estudiadas, cuenta Cetro coincidir cuenta Coulter. </li><li> Cuenta de 19 líneas celulares se realizó con un Z2 Coulter Counter, el contador celular Scepter, un contador automatizado basado en visión, como Vi-celular o sistema, comercios, y un hemocytemeter. </li><li> Los conteos se realizaron de acuerdo a las instrucciones del fabricante, utilizando la misma célula de partida suspensión y las diluciones idénticos. Los resultados mostrados en la Figura 3 demuestran que las concentraciones de células medida por cetro contar asemejan las concentraciones teóricas de células con una alta linealidad. </li><li> El coeficiente de variación por ciento, o CV, se ha calculado para cada dilución de cada línea celular para cada sistema de conteo. La Figura 4 muestra el CV% promedio de más de 19 líneas de células con respecto a la concentración de células y el método de recuento. </li><li> Dentro del rango de funcionamiento, el contador de células cetro es más preciso que el recuento del hemocitómetro visión basada, mostrando pequeñas desviaciones estándar y pequeños coeficientes promedio de la variación. </li><li> Contar cetro es diez veces más rápido que hemocytometry y más rápido que otros contadores automatizados también. En la figura 5, SF9, MCF7, y las células HEK293 se contaron usando los métodos descritos en este video. </li><li> El tiempo medio necesario para medir la concentración de células fue grabada. Tenga en cuenta que hemocytometry se llevó a cabo una sola vez por cada muestra. </li><li> Cetro que hace las mediciones basadas en el tamaño celular. Así, cualquier cambio en la morfología celular puede ser capturado en el histograma. Culturas saludables presenta una distribución en forma de campana de Gauss agradable, mientras que las células enfermas, lisis, y provocar un cambio en el histograma a la izquierda (Figura 6, panel izquierdo). </li><li> Del mismo modo, cuando una población de células muertas el 50% se mezclaron con las células del 50% en vivo, los cambios también se observaron histograma (Figura 6, panel derecho). </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, Das Chandreyee, Janet L. Smith, Gil Sonia, y Johnston Gracia son empleados por Millipore, que produce los instrumentos utilizados en este artículo.

Comparar el desempeño del contador de células Cetro de los resultados de otros métodos de recuento, se concluye que este nuevo contador de mano, de células automatizado proporciona el recuento de células precisa, rápida y confiable sobre un amplio rango de operación. La funcionalidad de contar Cetro es el resultado de la tecnología de ingeniería de precisión incrustado en la punta del sensor y la instrumentación de contar sofisticado basado en el principio de Coulter. La calidad del desempeño sugiere Cetro contando como método para aumentar la velocidad y mejorar la reproducibilidad de los ensayos basados ​​en células. Su formato de mano permite que el tedio rápida sin derecho a contar desde la capilla de cultivo de células.

<table border="1"><tbody><tr><td> Nombre del Material </td><td> Empresa </td><td> Número de catálogo </td><td> Comentario </td></tr><tr><td> Cetro de mano 2,0 contador celular automático con 40 sensores micras Cetro </td><td> EMD Millipore </td><td> PHCC20040 </td><td></td></tr><tr><td> Cetro de mano 2,0 contador celular automático con 60 sensores Cetro m </td><td> EMD Millipore </td><td> PHCC20060 </td><td></td></tr><tr><td> Sensores cetro, 60 micras </td><td> EMD Millipore </td><td> PHCC60050 </td><td> 50 unidades de sensores </td></tr><tr><td> Sensores cetro, 40 m </td><td> EMD Millipore </td><td> PHCC40050 </td><td> 50 unidades de sensores </td></tr><tr><td> Adaptador universal de energía </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 anillos y una tapa del filtro </td></tr></tbody></table>

determining cell number during cell culture using the scepter cell counter

El recuento de células es a menudo un paso necesario, pero tedioso para cultivo celular in vitro. Concentraciones consistentes celular asegurar la reproducibilidad experimental y precisión. Los recuentos de células son importantes para la vigilancia de la salud celular y la tasa de proliferación, la evaluación de la inmortalización y transformación, la siembra de células de experimentos posteriores, transfección o infección, y la preparación de ensayos basados ​​en células. Es importante que el recuento de células se precisa, coherente y rápida, en particular para la medición cuantitativa de la respuesta celular. A pesar de esta necesidad de la velocidad y la precisión en el recuento de células, el 71% de los 400 investigadores encuestaron a una que cuentan las células utilizando un hemocitómetro. Mientras hemocytometry es barato, es laborioso y sujeto a un sesgo de usuario y el uso indebido, que se traduce en cuenta inexacta. Hemocytometers están hechas de vidrio óptico especial en el que las suspensiones celulares se cargan en los volúmenes especificados y contados bajo un microscopio. Fuentes de errores en hemocytometry incluyen: la distribución desigual de células en la muestra, de más o de muy pocas células en la muestra, las decisiones subjetivas en cuanto a si una célula dada entra en el área de conteo definido, la contaminación del hemocitómetro, de usuario a usuario variación , y la variación de la tasa de llenado de 2 hemocitómetro. Para aliviar el tedio asociado con el conteo manual, el 29% de los investigadores cuentan con las células de células automatizado contar los dispositivos, que incluyen la visión basada en los contadores, sistemas que detectan las células utilizando el principio de Coulter, o citometría de flujo 1. Para la mayoría de los investigadores, la principal barrera a la utilización de un sistema automatizado es el precio asociado con estos grandes instrumentos de sobremesa 1. El contador de células cetro es un dispositivo automático de mano que ofrece la automatización y la exactitud del conteo Coulter, a un costo relativamente bajo. El sistema utiliza el principio de la detección de partículas Coulter impedancia basada en tres en un formato de miniatura utilizando una combinación de hardware analógico y digital para la detección, procesamiento de señales, almacenamiento de datos y visualización gráfica. La punta desechable se ha diseñado con un microfabricated, células de detección de la zona que permite la discriminación por tamaño de la celda y el volumen celular a nivel sub-micrones y la resolución de sub-picolitros. Mejorado con una precisión de manejo de líquidos de los canales y la electrónica, el contador de células Cetro informes de estadísticas de la población de células muestran gráficamente en forma de 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

El contador de células cetro es un dispositivo portátil automatizado que puede ser usado para contar las células, monitor diámetro de la celda y el volumen, y ser usado para controlar la salud y la calidad de las poblaciones celulares de una cultura a otra.

Determinar el número de células durante el cultivo celular utilizando el contador de células Cetro

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 vistas.  Millipore Inc. El contador de células cetro es un dispositivo portátil automatizado que puede ser usado para contar las células, monitor diámetro de la celda y el volumen, y ser usado para controlar la salud y la calidad de las poblaciones celulares de una cultura a otra.

Video: 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 madre embrionarias humanas: 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  ...

Investigación

Biología

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.

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

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.

Lentiviral mediada por derribo Durante Ex Vivo Eritropoyesis de las células estaminales hematopoyéticas

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.

Determinación del recuento de células de mamíferos, el tamaño celular y la salud de la célula mediante el Moxi Z Mini contador automatizado de células

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.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

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.

Los explantes blastómeras para la Prueba de Compromiso destino celular durante el desarrollo embrionario

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.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

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.

Comparación de tres diferentes métodos para determinar la proliferación celular en líneas celulares de cáncer de 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.

Vivo de la célula de microscopía de fluorescencia para observar procesos esenciales durante el crecimiento de la célula microbiana

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.