This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

Los experimentos con muestras de sangre se realizaron en cumplimiento de las leyes y directrices institucionales. Las muestras fueron las muestras de sangre residual que habían sido adquiridas y procesadas en los ensayos clínicos en la institución. 1. PT-AS Configuración óptico NOTA: Se puede utilizar un tubo de micro-capilar de vacío para una configuración inicial PT-AS. <ol><li> Montar un tubo de micro-capilar vacío con diámetros interior y exterior de 200 y 330 micras, respectivamente, y una longitud de más de ~ 5 cm en un accesorio de tubo capilar. accesorios de fibra comercialmente disponibles se pueden usar como el accesorio de tubo. </li><li> Segura anclar un puntero láser de 650 nm, es decir, la sonda fuente de luz, para iluminar el tubo capilar. El haz de sonda debe ser mayor que el tubo capilar. Coloque una pantalla (por ejemplo, papel blanco) por detrás del tubo capilar para observar un patrón periódico angular. </li><li> Para la parte de detección, quitar los lentes de una cámara web para capturar directamente el scattEring patrón. Coloque la cámara detrás del tubo capilar en un ángulo de 25-35 ° con respecto a la dirección del haz de la sonda. Asegúrese de que el patrón periódico angular producido por el tubo capilar se puede medir con el detector (Figura 1). Observe el patrón periódico angular en el medio del sensor de imagen cuando el sensor de imagen está colocada adecuadamente. </li><li> Posicionar una fuente de luz de excitación PT-532 nm para iluminar el tubo capilar. Coloque la fuente de luz PT en cualquier ángulo, siempre y cuando la luz de excitación se superpone con PT haz de sonda en el tubo capilar y no llega al detector directamente. excitación PT de muestras de sangre usando alta potencia óptica típicamente mejora la PT-AS sensibilidad, ya que conduce a un cambio mayor en la RI. <ol><li> Utilice la potencia óptica más alta de la fuente de luz de excitación PT empleado. Además, asegúrese de que la luz de excitación PT se superpone a la luz de la sonda en el tubo capilar. Utilice un tamaño de haz de la luz de excitación PTal menos dos veces la de la luz de la sonda para calentar todo el volumen de la sonda. </li></ol></li><li> Coloque un filtro de paso largo en frente del detector para bloquear la luz de 532 nm y medir sólo la luz de la sonda de 650 nm. </li><li> Instalar una cuchilla óptica en la trayectoria de la luz de excitación PT antes de iluminar el tubo capilar. La cuchilla óptica se emplea para modular la intensidad PT luz de excitación. </li></ol> Preparación de la muestra 2. Blood <ol><li> Dibujar 6 ml de sangre total fresca en estado anémico en tubos de muestras de sangre de ácido etilendiaminotetraacético, y mezclar bien las muestras. No se requiere ningún otro proceso. </li><li> Medir las muestras de sangre utilizando el sensor PT-AS dentro de 24 h de extracción para evitar la coagulación. </li></ol> 3.-AS PT Protocolos de medición <ol><li> Cargar un tubo de micro-capilar con una muestra de sangre para medir. Llenar el tubo capilar con la sangre a través de la acción capilar al colocar el tubo en el s sangreamplio. El volumen mínimo de muestra requerido para la medición se determina por el diámetro interior del tubo capilar y el tamaño del haz de la sonda. <ol><li> Emplear un tubo con un diámetro interior de 200 micras. El tamaño del haz de la sonda fue de 2 mm en los resultados representativos, lo que sugiere que la medición puede realizarse con un volumen de muestra de&gt; 63 nl. </li></ol></li><li> Montar el tubo capilar en la posición designada en el accesorio. </li><li> Encienda el láser sonda de 650 nm para iluminar el tubo capilar micro-cargado de sangre. El patrón periódico angular debe ser observado con la cámara web. </li><li> Encienda el 532-nm láser de excitación PT para iluminar el tubo. </li><li> Ejecutar la cuchilla óptica para modular la intensidad de la luz de excitación PT a 2 Hz. NOTA: La justificación de la selección de este estado de funcionamiento se describe en Discusión y Kim et al. 12. <ol><li> Montar una rueda perforada en el conjunto de la cabeza del motor de la cuchilla ópticasistema. </li><li> Encienda la caja de control del chopper, y utilice el mando de control en la consola para configurar la frecuencia de modulación. </li><li> Ejecutar el helicóptero con el mando de control. </li></ol></li><li> Registrar el patrón de dispersión fluctuante a través de la cámara web durante 5 segundos en formato MPEG-4 (MP4). </li></ol> 4. Procesamiento de Señales NOTA: procesamiento de señales PT-AS se ha realizado mediante un código MATLAB desarrolladas en laboratorio. <ol><li> Cargar el archivo de vídeo para extraer las imágenes. Para cada imagen [véase la figura 2 (a) para una imagen representativa], obtener el patrón de dispersión promediado mediante el cálculo de la media de los valores de los píxeles a lo largo de la dirección vertical [Figura 2 (b, c)]. </li><li> Evaluar la transformada de Fourier del patrón de dispersión de promediado, y calcular la fase en la frecuencia espacial de pico. Realizar estas operaciones para todos los marcos de todas las imágenes grabadas. </li><li> Utilizando los valores de fase obtenidos de todas las imágenes, trazar la fase temporalfluctuación [Figura 2 (d)]. Tenga en cuenta que la fase fluctúa en la frecuencia de modulación PT. Tomar la transformada de Fourier de la fluctuación de fase en el dominio del tiempo, y la obtención de la magnitud en la frecuencia de modulación. Esta señal se conoce como el PT, como señal de [Figura 2 (e)]. </li><li> Medir la [Hb] de una muestra de sangre mediante la conversión de su señal de PT-AS en el correspondiente [Hb] utilizando la curva de calibración que se obtiene en el Protocolo 5. </li></ol> 5. PT-AS calibración <ol><li> Preparar muestras de sangre, que tiene [Hb] valores que se distribuyen de manera uniforme en el rango de detección del sensor PT-AS (por ejemplo, 0 a 18 g / dl). </li><li> Antes de la calibración, cuantificar los valores de [Hb] de las muestras usando un analizador de hematología de referencia. Medir las señales PT-A partir de las muestras. </li><li> Derivar una curva de calibración correspondiente [Hb] para la señal de PT-AS mediante la realización de un lineal de mínimos cuadrados encajan, [Hb] = A [PT-AS señal] + B, de los Experimental resultados. Para las condiciones de funcionamiento especificadas en la Tabla 1, se encontró que la relación entre la señal PT-AS [Hb] y ser [Hb] = 5,13 [señal de PT-AS] - 0,09. Utilice el código de MATLAB para realizar el ajuste lineal. </li></ol>

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No conflict of interest is declared.

El sensor PT-AS representa un método totalmente óptico capaz de medir directamente la [Hb] de las muestras de sangre no procesada. El método cuantifica [Hb] en la sangre usando la respuesta PT intrínseca de las moléculas de hemoglobina en los eritrocitos. Bajo iluminación con luz de 532 nm, las moléculas de Hb absorben la energía de la luz y producen calor. El aumento de temperatura resultante cambia el RI de la muestra de sangre. La alta sensibilidad de RI de BSI fue explotada para medir este cambio de RI en la...

Un análisis de sangre se realiza con frecuencia para evaluar la salud humana en general y para la detección de biomarcadores relacionados con ciertas enfermedades. Por ejemplo, la concentración de colesterol en la sangre sirve como criterio para la hiperlipidemia, que está estrechamente relacionada con enfermedades cardiovasculares y pancreatitis. El contenido de glucosa en sangre deben ser medidos con frecuencia, ya que el nivel de glucosa se asocia con complicaciones tales como la cetoacidosis diabética y el síndrome hiperosmolar hiperglucémico. Las enfermedades graves como la malaria, el virus de la inmunodeficiencia humana y el síndrome de inmunodeficiencia adquirida se diagnostica mediante exámenes de sangre, y la cuantificación de los componentes de la sangre incluyendo eritrocitos, trombocitos, leucocitos y permite la detección de enfermedades pancreáticas y renales. La hemoglobina (Hb), un componente crítico de la sangre, representa aproximadamente el 96% de los eritrocitos, y transporta oxígeno a los órganos humanos. alteración significativa de su concentración en masa ([Hb]) me puede indicarcambios tabólica, enfermedad hepatobiliar y trastornos neurológicos, cardiovasculares y endocrinológicas 1. Por lo tanto, [Hb] se mide de forma rutinaria en los análisis de sangre. En particular, los pacientes con anemia, los pacientes de diálisis, y las mujeres embarazadas se recomiendan fuertemente para vigilar [Hb] como una tarea fundamental 2. Así, se han desarrollado diversos métodos de detección [Hb]. El método de la hemoglobina cianuro, una de las técnicas más comunes para [Hb] cuantificación, emplea cianuro de potasio (KCN) para destruir la bicapa lipídica de los eritrocitos 3. La hemoglobina cianuro producido por el exposiciones químicas de alta absorción alrededor de 540 nm; Así pues, [Hb] mediciones pueden realizarse a través de análisis colorimétrico. Este método es ampliamente empleado debido a su simplicidad, pero los productos químicos empleados (por ejemplo, KCN y óxido de dimethyllaurylamine) son tóxicos para los humanos y el medio ambiente. El esquema de hematocrito mide la relación en volumen de células rojas de la sangre en comparación con el total de la sangre volume través de la separación centrífuga; sin embargo, requiere un volumen de sangre relativamente grande (50 a 100 l) 4. Espectrofotometría de métodos de medida [Hb] precisión, sin ningún producto químico, pero las mediciones de múltiples longitudes de onda y un volumen grande de sangre se requieren 5,6. Del mismo modo, se han propuesto varios métodos ópticos para la medición de [Hb] incluidos los métodos de detección basados ​​en dispersión de luz, pero sus exactitudes de medición dependerá en gran medida de la precisión del modelo de sangre teórico. Para superar estas limitaciones, recientemente se han propuesto métodos de detección [Hb] basados en el efecto fototérmica (PT) de Hb 7. Hb, que está compuesta principalmente de óxidos de hierro, absorbe la luz a 532 nm y convierte la energía de la luz en calor 8-10. Este aumento de temperatura PT puede detectarse ópticamente mediante la medición de un cambio en el índice de refracción (RI) de muestras de sangre. Yim et al. empleada dominio espectral de coherencia óptica reflectometry para medir el PT cambio de la longitud del recorrido óptico en una cámara 11 que contiene sangre. Aunque el método permite la medición directa [Hb] libre de productos químicos y, el uso de un espectrómetro y una disposición interferométrico pueden obstaculizar su miniaturización. Recientemente hemos presentado un método alternativo de detección [Hb], denominado sensor de foto-térmica angular de dispersión de luz (PT-AS), que es más adecuado para la miniaturización del dispositivo 12. El sensor PT-AS explota la alta sensibilidad RI de la interferometría de retrodispersión (BSI) para medir los cambios de PT de la RI de una muestra de sangre dentro de un tubo capilar. BSI se han utilizado para medir RI de diversas soluciones de 13-15 y para monitorear las interacciones bioquímicas en solución libre 16. El sensor PT-AS emplea a disposición óptica similar a la del BSI, pero combina la configuración de excitación fototérmica para medir el aumento de PT RI en muestras de sangre. Principios de funcionamiento de la BSI y los sensores PT-AS se describen en detalle en otra parte <sup&gt; 12,15. PT-AS sensor demostró alta sensibilidad de medición [Hb] en un amplio rango de detección (0,35 a 17,9 g / dl) y es capaz de funcionar con volúmenes de muestra de &lt;100 nl. No se requiere un preacondicionamiento de la muestra de sangre, y el tiempo de medición es solamente ~ 5 seg. A continuación, se describen la configuración experimental y un protocolo de medición detallada. Los resultados representativos PT-AS se proporcionan usando muestras de sangre de pacientes anémicos, y los resultados se comparan con los de un analizador de hematología para evaluar la precisión del sensor de PT-AS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>650nm laser pointer</td>
			<td>LASMAC</td>
			<td>LED-1</td>
			<td>Probe light</td>
		</tr>
		<tr>
			<td>Hollow round glass capillaries</td>
			<td>Cm Scientific</td>
			<td>CV2033</td>
			<td>Blood sample container</td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>C525</td>
			<td>CMOS optical sensor</td>
		</tr>
		<tr>
			<td>532-nm DPSS laser</td>
			<td>CNI Laser</td>
			<td>MGL-&#8546;-532</td>
			<td>Photothermal light source</td>
		</tr>
		<tr>
			<td>Optical chopper system</td>
			<td>Thorlabs</td>
			<td>MC2000-EC</td>
			<td>Optical chopper</td>
		</tr>
		<tr>
			<td>Plastic long-pass filter</td>
			<td>Edmund Optics</td>
			<td>#43-942</td>
			<td>To reject 532-nm PT excitation light</td>
		</tr>
		<tr>
			<td>Fiber clamp</td>
			<td>Thorlabs</td>
			<td>SM1F1-250</td>
			<td>Capillary tube fixture</td>
		</tr>
		<tr>
			<td>EDTA coated blood sampling tube</td>
			<td>Greiner Bio-One</td>
			<td>VACUETTE 454217</td>
			<td>Blood sampling &#38; anticoagulating</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Siemens AG</td>
			<td>ADVIA 2120i</td>
			<td>Reference hematology analyzer</td>
		</tr>
	</tbody>
</table>

a rapid and chemical-free hemoglobin assay with photothermal angular light scattering

Photo-térmica angular de dispersión de luz (PT-AS) es un método óptico novedoso para la medición de la concentración de hemoglobina ([Hb]) de muestras de sangre. Sobre la base de la respuesta fototérmica intrínseca de las moléculas de hemoglobina, el sensor permite una alta sensibilidad, la medición libre de químicos de [Hb]. [Hb] capacidad de detección con un límite de 0,12 g / dl en el rango de 0,35 a 17,9 g / dl se ha demostrado previamente. El método se puede implementar fácilmente utilizando dispositivos electrónicos de bajo costo de consumo, tales como un puntero láser y una cámara web. El uso de un tubo de micro-capilar como un recipiente de sangre también permite el ensayo de la hemoglobina con un volumen de sangre de nanolitros escala y un bajo costo de operación. A continuación, se presentan instrucciones detalladas para los procedimientos de procesamiento de configuración PT-ópticos y de señal. También se proporcionan los protocolos experimentales y los resultados representativos de las muestras de sangre en condiciones anémicas ([Hb] = 5,3, 7,5, y 9,9 g / dl), y las mediciones se comparan con los froanalizador de hematología ma. Su simplicidad en la implementación y operación de permitir su adopción generalizada en los laboratorios clínicos y entornos con recursos limitados.

Un ensayo de hemoglobina se realizó utilizando el sensor PT-AS, y sus mediciones se compararon con los de un analizador de hematología. El experimento se realizó con una intensidad de luz de excitación PT de 1,4 W / cm 2, PT frecuencia de modulación de 2 Hz, y la medición de tiempo de 5 seg. La Tabla 1 resume las condiciones experimentales. Los tamaños de haz de la sonda y la excitación de la luz PT fueron del 5,5 y 2 mm, respectivamente. La cámara we...

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A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Un ensayo de hemoglobina rápida y libre de químicos con fototérmica angular de dispersión de luz

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

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9.6K vistas.  Yonsei University. A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Video: A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

Patrones sustractiva rápida de las capas de células vivas con una sonda de microfluidos

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Investigación

Bioingeniería

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formación luz mediada y el patrón de hidrogeles para aplicaciones de cultivo celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Mezcla rápida Preparación de Bioinspirada nanoescala hidroxiapatita para aplicaciones biomédicas

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

Construcción y configuración de un biorreactor fotosintético de algas a escala de banco con control de temperatura, luz y pH para pruebas de crecimiento cinético

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

Protocolo para la transferencia de microARN en médula ósea hematopoyéticas células madre adultas para permitir ingeniería celular combinado con orientación magnética

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Un análisis de la virulencia bacteriana rápida basada en imágenes usando ameba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

Caracterización rápida de partes genéticas con sistemas libres de células

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Fermentaciones controladas por la luz para la producción química microbiana y de proteínas

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

In vivo Imágenes de tejidos biológicos con fluorescencia combinada de dos fotones y microscopía de dispersión Raman estimulada

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

Un enfoque no secuenciador para la detección rápida de la edición de ARN

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...