Nos gustaría agradecer a la Fundación Canadiense para la Innovación, el British Columbia Fondo de Desarrollo de conocimientos y el Nacional de Ciencias e Ingeniería de Investigación (NSERC) de Canadá para apoyar los estudios en curso en las regiones de bajo nivel de oxígeno de las aguas oceánicas costeras y abiertas. DAW fue apoyado por becas de NSERC, Killam y la Fundación Centro TULA financiado por la diversidad microbiana y la evolución.

<ol><li> Puesta en marcha de aparatos de filtración <ol><li> Para configurar el equipo de filtración, use pinzas para colocar un grado GF / D de filtro en la unidad de vivienda pre-filtro. Apriete ligeramente la carcasa con la mano. Como regla general, no apretar en exceso o de la unidad se fuga una vez que se inicia el bombeo. </li><li> Vuelva a colocar la tapa de la bombona con la tapa del autoclave y tubos conectados por cuidado de insertar el tubo interno en la bombona, pero sin tocarlo. Si es necesario, use pinzas para guiar el tubo en la garrafa. </li><li> Pase la tubería externa a través de la bomba peristáltica. </li><li> Colocar las unidades de vivienda prefiltro hasta el final de los dos tubos externos (es decir, 2 unidades por bombona). Apriete la conexión con los alicates. </li></ol></li><li> Filtración de agua de mar <ol><li> Para empezar a filtrar el agua de mar, a su vez en la bomba y ajustar la velocidad de flujo a 1. Reloj para asegurar que el agua se bombea en la dirección correcta. </li><li> Permita que 500 ml de agua a fluir libremente a través de la unidad de pre-filtro y en un vaso de 1 litro. </li><li> Apague la bomba y coloque un filtro Sterivex a la conexión luer macho en el extremo libre de la configuración del prefiltro. Apriete el prefiltro de la vivienda cómodamente con la mano. </li><li> Esté atento a la vivienda en el prefiltro y la conexión entre la vivienda pre-filtro y el tubo que conduce a la bombona. Si se producen fugas, apagar la bomba y aflojar y apretar la cubierta prefiltro en consecuencia. Encienda la bomba de nuevo. </li><li> Recoger el filtrado en 4 frascos L. Una vez que se llena y vacía los frascos en una bombona de limpieza. Asegúrese de anotar el volumen pasa a través de cada filtro. Recogidos filtrado pueden ser procesados, tales como la filtración de virus. </li><li> Continuar el filtrado hasta que no quede agua en el bombonas. Filtración habitual gama veces entre 4-5 h / 20 bombona L, sin embargo, esto depende de manera dramática en la cantidad de biomasa en el agua. </li><li> Una vez que la filtración se haya completado, el interruptor de la bomba y desconecte el filtro Sterivex. </li></ol></li><li> El almacenamiento de filtros <ol><li> Para almacenar los filtros, en primer lugar expulsar cualquier resto de agua con una jeringa de 30-60. </li><li> Con una pipeta, añadir 1,8 ml de solución amortiguadora de lisis del filtro, manteniendo ~ 200 ul de espacio en el filtro para la adición posterior de los reactivos. </li><li> Selle la parte inferior y superior del filtro Sterivex con un pequeño trozo de Parafilm, y la etiqueta de los filtros con la fecha y la identificación de la muestra. </li><li> Tienda de los filtros en un pre-etiquetados tubo Falcon de 50 ml a -80 ° C </li><li> Retire los filtros de la unidad de vivienda y colocarlos en un recipiente etiquetado tubos Falcon de 50 ml que contiene 2 ml de tampón de lisis. Guarde estos documentos a -80 ° C. </li></ol></li><li> Limpieza <ol><li> Para la limpieza final, coloque el tubo que estaba dentro de la bombona en un matraz que contenía 0,1 M de HCl </li><li> Dirigir la tubería que conducen al matraz 4L en la bombona original. </li><li> Encienda la bomba y correr a los 3, recogiendo el flujo a través de carreras en la bombona original. </li><li> Por último, enjuagar la tubería con agua esterilizada. </li></ol></li></ol> Los resultados representativos: El resultado final consiste en dos filtros Sterivix cada uno con la biomasa microbiana del agua de mar de aproximadamente 10 L en el rango de tamaño de 0.22μm y 2.7μm. Esto se almacena en buffer de lisis y listo para la extracción de ADN. Además, este procedimiento se acumula y concentra la biomasa superior a 2,7 micras, en los prefiltros GF / D. Esto también se almacena en buffer de lisis y listo para la extracción de ADN. Si los pasos de procesamiento adicionales se toman, el 0,22 M a través de flujo se pueden concentrar hasta recoger la fracción viral también.

The authors have nothing to disclose.

Es imprescindible para limpiar los bancos y las bombas con una toalla húmeda después de cada uso para eliminar cualquier rastro persistente de agua salada que de otra manera corroer el equipo. Aunque el procedimiento es simple, el tiempo requerido puede ser extensa en función de la concentración de la biomasa o los residuos de la muestra que se filtra.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Peristaltic pump</td>
<td>&nbsp;</td>
<td>Masterflex (Cole Palmer)</td>
<td>77913-20, 77200-62, 77200-04</td>
<td>&nbsp;</td>
<tr>
<td>Tygon LFL tubing</td>
<td>&nbsp;</td>
<td>Masterflex (Cole Palmer)</td>
<td>06429-24</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Hose clamps</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>0683204</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>1/4&#x201D; x 3/16&#x201D; pipe adaptors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>31702-20</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Polypropylene prefilter housing</td>
<td>&nbsp;</td>
<td>ADVANTEC</td>
<td>501200</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Metal luer fitting (Hose end to Male luer lock)</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>31507-27</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>2&#x201C; Magnets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For filtration apparatus</td>
<td>&nbsp;</td>
<tr>
<td>3 port carboy caps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>GV 0.22 &mu;m STERIVIX filters</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SVGV01R5</td>
<td>&nbsp;</td>
<tr>
<td>47 mm diameter 2.7 &mu;m GF/D filters</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1823047</td>
<td>&nbsp;</td>
<tr>
<td>Lysis buffer</td>
<td> Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3)</td>
</tr>
<tr>
<td>0.1 % HCl</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For post-filtration rinse</td>
</tr>
<tr>
<td>Autoclaved water</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For post-filtration rinse</td>
</tr>		</tbody>
		</table>
		</div>

large volume (20l+) filtration of coastal seawater samples

El flujo de trabajo comienza con la recolección de las aguas marinas costeras de la comunidad microbiana abajo, análisis de gases en nutrientes y oligoelementos. Para este método, se recogieron muestras de la cubierta de la operación HMS John Strickland en Saanich Inlet. Este volumen de vídeo documentos de gran tamaño (≥ 20 L) de filtración de la biomasa microbiana, que oscilan entre 0.22μm y 2.7μm de diámetro, desde la columna de agua. Dos muestras de 20 litros se pueden filtrar de forma simultánea utilizando una unidad de una sola bomba equipada con cuatro cabezales rotativos. La filtración se lleva a cabo en el campo de los viajes largos, o inmediatamente después del regreso de excursiones de un día. Es importante registrar la cantidad de agua que pasa por cada unidad sterivex filtro. Para prevenir la formación de biofilm entre los viajes de muestreo, todo el equipo de filtración debe ser enjuagado con HCl diluido y agua destilada y autoclave inmediatamente después del uso. Este procedimiento durará aproximadamente cinco horas más una hora adicional para la limpieza.

Watch this Scientific Journal Video about Large Volume 20L+ Filtration of Coastal Seawater Samples at JoVE.com

Large Volume 20L+ Filtration of Coastal Seawater Samples

Este volumen de vídeo documentos de gran tamaño (≥ 20 L) de filtración de la biomasa microbiana, que oscilan entre 0.22μm y 2.7μm de diámetro, desde la columna de agua.

De gran volumen (20 litros +) Filtración de muestras de agua marina costera

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

large-volume-20l-filtration-of-coastal-seawater-samples

Research

JoVE Journal

Biology

Large Volume (20L+) Filtration of Coastal Seawater Samples

11.7K vistas.  University of British Columbia - UBC. Este volumen de vídeo documentos de gran tamaño (≥ 20 L) de filtración de la biomasa microbiana, que oscilan entre 0.22μm y 2.7μm de diámetro, desde la columna de agua.

Video: Large Volume 20L+ Filtration of Coastal Seawater Samples

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

A gran escala de pantallas de bibliotecas metagenómicas

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Investigación

Biología

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

El agua de mar de muestreo y recolección

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

Volumen pequeño (1-3L) Filtración de muestras de agua marina costera

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Inserte grandes Ambiental Genómica Producción Biblioteca

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

La cuantificación de γH2AX focos en muestras de tejido

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absoluta Quantum medición del rendimiento de las muestras de polvo

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

La translocación de péptidos en la medición de grandes vesículas unilamelares

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Un método de alto rendimiento para la medición de la tasa de filtración glomerular en ratones conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Ensayo Infinium para aplicaciones a gran escala de genotipado de SNP

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Aislamiento rápido y purificación de mitocondrias para el trasplante por disociación de tejidos y filtración diferencial

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...