We thank J. Schmidt from the University of Freiburg for constructing the device. We thank P. Nielsen and D. Medgyesi for their support and critical reading of this manuscript. This study was supported by the Deutsche Forschungsgemeinschaft through SFB746 and by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

1. El diseño y la construcción del artificio <ol><li> Los experimentos piloto NOTA: La intensidad de la luz requerida para un tipo de herramienta y células optogenético específico puede variar significativamente. Los experimentos piloto con prototipos son útiles para estimar la intensidad de luz mínima necesaria. La herramienta optogenético utilizado para los siguientes experimentos es una construcción de fusión Dronpa-Enlazador Dronpa. Se obtuvo la secuencia comercialmente Dronpa (Ver Lista de Materiales). A largo enlazador se clonó en entre dos secuencias Dronpa para permitir que el constructo expresado para formar dímeros intramoleculares (e intermoleculares). Los pasos descritos a continuación se pueden adaptar a muchas otras herramientas optogenética o sustancias ópticamente controladas. <ol><li> Transducir los linfocitos B Ramos con un constructo que contiene Dronpa-Enlazador Dronpa siguiendo las instrucciones del fabricante para la transducción utilizando el sobrenadante que contiene retroviral de células de empaquetamiento. Cosecha y celdas de conteo de acuerdo con un prepublicada riormente protocolo 13. </li><li> Resuspender las células a una concentración de 1 x 10 6 / ml en medio (RPMI 1640, 10% FCS inactivado por calor, 2 mM L-glutamina, 100 U / ml de penicilina, 100 U / ml de estreptomicina y 50 mM 2-mercaptoetanol) y transferirlos a un tubo de FACS de vidrio. </li><li> Insertar el tubo en un citómetro de flujo y medir la intensidad de fluorescencia de Dronpa (canal GFP) 14,15. </li><li> Utilice gafas de protección para todas las medidas adicionales para proteger los ojos de longitudes de onda posiblemente perjudiciales. </li><li> colocar manualmente una luz LED 500 nm en el exterior del tubo y continuar midiendo la intensidad de fluorescencia de Dronpa. </li><li> Aumentar el número y / o la intensidad de las luces LED hasta que la intensidad de fluorescencia Dronpa disminuye. </li><li> colocar manualmente una luz LED 400 nm en el exterior del tubo y continuar midiendo la intensidad de fluorescencia de Dronpa. </li><li> Aumentar el número y / o la intensidad de las luces LED hasta que el fluo Dronpacencia aumenta la intensidad. </li><li> Utilice al menos tantas luces LED para la construcción del dispositivo como se prevé que sea necesario desde el photoswitching en los pasos 1.1.5 y 1.1.7. </li></ol></li><li> La construcción del dispositivo NOTA: El dispositivo fue construido por un taller de mecanizado profesional, el Arbeitsgruppe Technik de la Universidad de Friburgo. La mayoría de las piezas son hechas a medida y no está disponible comercialmente. Las dimensiones exactas de cada parte se representan en las figuras 1 y 2. Para aclarar el montaje de este dispositivo, complementaria de vídeo 1 se proporciona y los pasos más esenciales se describe a continuación. <ol><li> Molino pieza A partir de cloruro de polivinilo (PVC) con las medidas exactas que se muestran en la Figura 1A. </li><li> Inserte las luces LED en los agujeros perforados de pieza A y cerrar cada agujero con pegamento para PVC para evitar fugas de agua. </li><li> Conectar los LED a un transformador multicanal (cada longitud de onda debe ocupar un chann separadael) con una potencia de salida ajustable de 0-29 mA. </li><li> Coloque la cubierta exterior (B) con 3 tornillos a la pieza A como se representa para proteger el LED contra daño físico. </li><li> Para poder conectar el dispositivo a una bomba de agua, pegamento en los clips de tubo (C) en los agujeros perforados de pieza A (representada en la figura 1a, la sección transversal Y: 9 mm y 61,5 mm) con pegamento de PVC. </li><li> Cortar un largo tubo de plexiglás 58,5 mm (D1). </li><li> Fabricación piezas D2 y D3 de Plexiglas como se representa en la Figura 2. </li><li> piece pegamento D3 de la parte inferior de D1 con Plexiglas pegamento. </li><li> Adjuntar una junta tórica de caucho a la pieza D2 y péguelo en la parte superior de D1 con plexiglás pegamento. NOTA: Las piezas D1, D2 y D3 juntos forman pieza D. </li><li> Insertar pieza D en la pieza A con 3 tornillos. </li><li> Pruebe el dispositivo para la salida del agua antes de suministrar energía al transformador. </li></ol></li></ol> 2. Medición de la cinética de una herramienta Optogenética <ol&gt; <li> preparar las células <ol><li> Cultura células Ramos B en RPMI-medio (RPMI 1640, 10% FCS inactivado por calor, 2 mM L-glutamina, 100 U / ml de penicilina, 100 U / ml de estreptomicina y 50 mM 2-mercaptoetanol) a una densidad de 3- 10 x 10 6 células / ml a 37 ° C y 5% de CO2. </li><li> Cosecha Ramos B células por centrifugación a 350 xg y 4 ° C durante 5 min. </li><li> Recuento de células utilizando una cámara de recuento Neubauer siguientes instrucciones del fabricante y resuspender 1-5 x 10 6 células en 600 l de medio. </li><li> Transferir las células a un tubo de vidrio de FACS, ponerlos en hielo y protegerlos de la luz hasta la medición. </li></ol></li><li> Preparar el citómetro de flujo y el dispositivo. <ol><li> Conectar el dispositivo a una bomba de agua caliente y ajustar la temperatura a 37 ° C. </li><li> Cuando se utilizan tubos de FACS vidrio, intercambiar el negro junta tórica del citómetro de flujo con una junta tórica de caucho y, además, aplicar gr de siliciofacilitar. </li></ol></li><li> Medición <ol><li> Introduzca con cuidado el tubo de FACS de vidrio en el dispositivo y conectarlo con el citómetro de flujo (Figura 3). </li><li> Iniciar la medición y registrar Dronpa intensidad fluorescente (GFP canal). </li><li> Iluminar la muestra de células con 400 nm o 500 nm de luz, respectivamente, y registrar la intensidad de fluorescencia Dronpa (canal GFP). </li></ol></li><li> Análisis de los datos <ol><li> Analizar los datos experimentales con flujo adecuado citómetro de software. Gráfico de dispersión hacia adelante contra la dispersión lateral y la puerta de las células vivas. Trazar la intensidad de fluorescencia Dronpa lo largo del tiempo. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

El flujo de Thermo LED es un dispositivo innovador para estudiar herramientas optogenética en un citómetro de flujo. Hasta el momento, las muestras han sido optogenética sólo iluminada con rayos láser o dispositivos de microscopía linterna 11,12. Dependiendo del ángulo y la distancia de la linterna a la muestra, se espera que la variabilidad sustancial en la cantidad de iluminación entre los experimentos. Además, hay un límite en el número de linternas una sola persona ...

Herramientas Optogenética han ido ganando popularidad, en parte debido a que se pueden utilizar para descifrar el cableado de las vías de señalización de 1-4. Se basan en la capacidad de las proteínas fotoactivables para cambiar su conformación y de unión por afinidad cuando se ilumina con la luz. La fusión de estas proteínas a los elementos de señalización permite la regulación específica de un solo jugador dentro de complejas vías de señalización intracelular 5-12. Por consiguiente, una vía de señalización puede ser estudiado con alta resolución temporal y espacial. La mayoría de los estudios optogenética basados en células utilizan métodos basados en la microscopía combinado con el cultivo en presencia de luz, seguido de análisis bioquímico 11,12. Por el contrario, un flujo singulariza citómetro de células a lo largo de un capilar y mide el tamaño celular, la granularidad y la intensidad de fluorescencia. Este método tiene ventajas importantes sobre microscopía o bioquímicos métodos, incluyendo la capacidad de analizar thousands de células a una resolución de una sola célula de vivir en un tiempo muy corto. Por lo tanto, es deseable combinar optogenética con citometría de flujo. Hasta donde sabemos, no existe un protocolo establecido para citometría de flujo optogenético. Un procedimiento ampliamente aceptado es iluminar manualmente las células desde el exterior del tubo de reacción con dispositivos de linterna. Sin embargo, la iluminación en el manual de flujo requiere citómetro de que la luz pase a través del tubo de reacción y, por imágenes de células vivas, una forma cilíndrica, cámara de agua caliente. Esto hace que la dispersión de luz y sustancial pérdida de luz. Por otra parte, la intensidad de la luz proporcionada por la iluminación manual no es reproducible entre experimentos (ángulo, distancia, etc.) y hay un límite práctico para el número de longitudes de onda en un experimento. Mediante la construcción del dispositivo de flujo Thermo LED, hemos sido capaces de superar estas limitaciones. Con este dispositivo, las células pueden ser iluminadas con longitudes de onda específicas en un tempratura controlada de forma durante las mediciones de citometría de flujo. Esto permite que las cantidades precisas y reproducibles de la luz dentro y entre los experimentos. Para demostrar la utilidad de nuestro dispositivo in vivo, se registró la señal de fluorescencia de Dronpa en las células B Ramos durante photoswitching. células Ramos B se derivan de un linfoma de Burkitt humano. Dronpa es una proteína fluorescente que existe como un monómero, dímero o tetrámero. En su forma monomérica, es no fluorescente. Iluminación con luz de 400 nm induce la dimerización y tetramerización y hace que la proteína fluorescente Dronpa. Este proceso puede ser revertido por iluminación con luz de 500 nm. La proteína Dronpa se ha utilizado para controlar la función y la ubicación de las proteínas de señalización de 4,13. Aquí, hemos expresado una proteína Dronpa engarce-Dronpa en las células B Ramos para estudiar photoswitching de Dronpa en un citómetro de flujo. El uso de nuestro dispositivo, hemos sido capaces de manera eficiente yreproducible PHOTOSWITCH Dronpa durante la grabación de su intensidad de fluorescencia en tiempo real. Este método ofrece ventajas sustanciales sobre los protocolos actuales de iluminación con la iluminación manual y amplía considerablemente el repertorio experimental para herramientas optogenética y compuestos jaula. El uso de nuestro dispositivo simplificará significativamente y acelerar el descubrimiento y desarrollo de nuevas herramientas optogenética.

<table border="0" cellpadding="0" cellspacing="0" width="1212">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass FACS tube</td>
			<td style="width:357px;">&#160;Thermo Fisher Scientific; Waltham, USA</td>
			<td style="width:312px;">14-961-26</td>
			<td style="width:296px;">Borosilicate glass tubes 12x75 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Flow Cytometer</td>
			<td style="width:357px;">BD Bioscienceg; Heidelberg, Germany</td>
			<td style="width:312px;">Fortessa II</td>
			<td>Special Order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dronpa: pcDNA3-mNeptune2-N</td>
			<td style="width:357px;">Addgene; Cambridge, USA</td>
			<td style="width:312px;">41645</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PolyJet</td>
			<td style="width:357px;">SignaGen, Rockville, USA</td>
			<td style="width:312px;">SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LED 505 nm</td>
			<td style="width:357px;">Avago Technologies; Boeblingen, Germany</td>
			<td style="width:312px;">HLMP-CE34-Y1CDD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LED 400 nm</td>
			<td style="width:357px;">Avago Technologies; Boeblingen, Germany</td>
			<td style="width:312px;">UV5TZ-400-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas tube 15 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">76999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 3 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 2,5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 1,5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC tile 5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC tile 6 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC block 50 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RPMI</td>
			<td style="width:357px;">Invitrogen, Life Technologies; Darmstadt, Germany</td>
			<td style="width:312px;">61870-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-Mercaptoethanol</td>
			<td style="width:357px;">EMD; Germany</td>
			<td style="width:312px;">805740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">FCS</td>
			<td style="width:357px;">PAN Biotech; Aidenbach, Germany</td>
			<td style="width:312px;">P30-3302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Penicillin/Strptomycin (10000 U/ml)</td>
			<td style="width:357px;">Invitrogen, Life Technologies; Darmstadt, Germany</td>
			<td style="width:312px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acrifix plexiglas glue</td>
			<td style="width:357px;">Evonic industries, Essen, Germany</td>
			<td style="width:312px;">1R0192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tangit PVC-U glue</td>
			<td style="width:357px;">Henkel, D&#252;sseldorf, Germany</td>
			<td style="width:312px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

led thermo flow &#8212; combining optogenetics with flow cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Utilizando el flujo de Thermo LED con un citómetro de flujo El núcleo funcional del dispositivo es una cámara cilíndrica en la que las luces LED están dispuestos en una forma circular que apunta hacia el interior. Esta cámara se puede conectar a un suministro de agua y la bomba, lo que permite controlar la temperatura de los LED, así como la muestra de células. Los LED están conectados a un transformador y, por tanto, l...

Watch this Scientific Journal Video about LED Thermo Flow â€” Combining Optogenetics with Flow Cytometry at JoVE.com

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Thermo LED Flujo - Combinando optogenética con Citometría de Flujo

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

led-thermo-flow-combining-optogenetics-with-flow-cytometry

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JoVE Journal

Biology

8.5K vistas.  Max-Planck Institute for Immunobiology und Epigenetics. Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Video: LED Thermo Flow — Combining Optogenetics with Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP-FCM: inmunoprecipitación detectada por citometría de flujo

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Investigación

Biología

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Citometría de flujo de purificación de las células de ratón meiótica

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Célula de medida Cinética la progresión del ciclo con el etiquetado metabólico y Citometría de Flujo

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Citometría de flujo basado Purificación de<em&gt; S. cerevisiae</em&gt; Los cigotos

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimizado manchas y Proliferación métodos de modelización para la vigilancia del uso de tintes División Celular Celulares de seguimiento

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Técnicas para el Análisis de extracelular vesículas Usando citometría de flujo

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análisis de suspensiones celulares aisladas de tejidos sólidos por Spectral Citometría de Flujo

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endorreduplicación medición por citometría de flujo de protoplastos aislados de tubérculos

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Medida de alto rendimiento de macropinocitosis discoideum de Dictyostelium por citometría de flujo

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...