Name: a strategy for sensitive, large scale quantitative metabolomics
Uploaded: 2014-05-27T13:45:00
Description: Watch this Scientific Journal Video about A Strategy for Sensitive, Large Scale Quantitative Metabolomics at JoVE.com

The authors would like to acknowledge Detlef Schumann, Jennifer Sutton (Thermo Fisher Scientific) and Nathaniel Snyder (University of Pennsylvania) for valuable discussions on mass calibration and data processing.&#160; Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R00CA168997. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Preparation of LC-MS Reagents, Establishment of a Chromatography Method, and Establishment of Instrument Operating Procedures
<ol>
	<li>Preparation of LC Solvents
	<ol>
		<li>Prepare 500 ml mobile phases. A is 20 mM ammonium acetate and 15 mM ammonium hydroxide in 3% acetonitrile/water, final pH 9.0;&#160;and B is 100% acetonitrile.</li>
		<li>Loosely cap the bottle, place it in a water bath sonicator, and sonicate for 10 min without extra heating. (This step is to ensure that all of the ammonium salts completely di...

<ol>
	<li>Fiehn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+genomics,+metabolome+analysis,+and+biochemical+modelling+to+understand+metabolic+networks.">Combining genomics, metabolome analysis, and biochemical modelling to understand metabolic networks.</a> Comparative and Functional Genomics. 2 (3), 155-168 (2001).</li><li>Mulleder, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prototrophic+deletion+mutant+collection+for+yeast+metabolomics+and+systems+biology.">A prototrophic deletion mutant collection for yeast metabolomics and systems biology.</a> Nature Biotechnology. 30 (12), 1176-1178 (2012).</li><li>Patel, V. R., Eckel-Mahan, K., Sassone-Corsi, P., Baldi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CircadiOmics:+Integrating+circadian+genomics,+transcriptomics,+proteomics+and+metabolomics.">CircadiOmics: Integrating circadian genomics, transcriptomics, proteomics and metabolomics.</a> Nature Methods. 9 (8), 772-773 (2012).</li><li>Ideker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+genomic+and+proteomic+analyses+of+a+systematically+perturbed+metabolic+network.">Integrated genomic and proteomic analyses of a systematically perturbed metabolic network.</a> Science. 292 (5518), 929-934 (2001).</li><li>Jonsson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+strategy+for+identifying+differences+in+large+series+of+metabolomic+samples+analyzed+by+GC/MS.">A strategy for identifying differences in large series of metabolomic samples analyzed by GC/MS.</a> Analytical Chemistry. 76 (6), 1738-1745 (2004).</li><li>Jonsson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+data+analysis+for+detecting+and+identifying+differences+between+samples+in+GC/MS-based+metabolomic+analyses.">High-throughput data analysis for detecting and identifying differences between samples in GC/MS-based metabolomic analyses.</a> Analytical Chemistry. 77 (17), 5635-5642 (2005).</li><li>Weljie, A. M., Newton, J., Mercier, P., Carlson, E., Slupsky, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+profiling:+Quantitative+analysis+of+1H+NMR+metabolomics+data.">Targeted profiling: Quantitative analysis of 1H NMR metabolomics data.</a> Analytical Chemistry. 78 (13), 4430-4442 (2006).</li><li>Wiklund, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+GC/TOF-MS-based+metabolomics+data+for+identification+of+biochemically+interesting+compounds+using+OPLS+class+models.">Visualization of GC/TOF-MS-based metabolomics data for identification of biochemically interesting compounds using OPLS class models.</a> Analytical Chemistry. 80 (1), 115-122 (2008).</li><li>Xia, J., Bjorndahl, T. C., Tang, P., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetaboMiner--semi-automated+identification+of+metabolites+from+2D+NMR+spectra+of+complex+biofluids.">MetaboMiner--semi-automated identification of metabolites from 2D NMR spectra of complex biofluids.</a> BMC Bioinformatics. 9, 507 (2008).</li><li>Lu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C+NMR+isotopomer+analysis+reveals+a+connection+between+pyruvate+cycling+and+glucose-stimulated+insulin+secretion+(GSIS).">13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS).</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (5), 2708-2713 (2002).</li><li>Shen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+rate+of+the+glutamate/glutamine+cycle+in+the+human+brain+b.+in+vivo+13C+NMR.">Determination of the rate of the glutamate/glutamine cycle in the human brain b. in vivo 13C NMR.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (14), 8235-8240 (1999).</li><li>Kitteringham, N. R., Jenkins, R. E., Lane, C. S., Elliott, V. L., Park, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+reaction+monitoring+for+quantitative+biomarker+analysis+in+proteomics+and+metabolomics.">Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics.</a> Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 877 (13), 1229-1239 (2009).</li><li>Locasale, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+of+human+cerebrospinal+fluid+identifies+signatures+of+malignant+glioma.">Metabolomics of human cerebrospinal fluid identifies signatures of malignant glioma.</a> Molecular &amp; Cellular Proteomics : MCP. 11 (6), 10-1074 (2012).</li><li>Wolf-Yadlin, A., Hautaniemi, S., Lauffenburger, D. A., White, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+reaction+monitoring+for+robust+quantitative+proteomic+analysis+of+cellular+signaling+networks.">Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (14), 5860-5865 (2007).</li><li>Yuan, M., Breitkopf, S. B., Yang, X., Asara, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+positive/negative+ion-switching,+targeted+mass+spectrometry-based+metabolomics+platform+for+bodily+fluids,+cells,+and+fresh+and+fixed+tissue.">A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue.</a> Nature Protocols. 7 (5), 872-881 (2012).</li><li>Ramanathan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=It+is+time+for+a+paradigm+shift+in+drug+discovery+bioanalysis:+From+SRM+to+HRMS.">It is time for a paradigm shift in drug discovery bioanalysis: From SRM to HRMS.</a> Journal of Mass Spectrometry : JM. 46 (6), 595-601 (2011).</li><li>Lu, W., Clasquin, M. F., Melamud, E., Amador-Noguez, D., Caudy, A. A., Rabinowitz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+analysis+via+reversed-phase+ion-pairing+liquid+chromatography+coupled+to+a+stand+alone+orbitrap+mass+spectrometer.">Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer.</a> Analytical Chemistry. 82 (8), 3212-3221 (2010).</li><li>Michalski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra+high+resolution+linear+ion+trap+orbitrap+mass+spectrometer+(orbitrap+elite)+facilitates+top+down+LC+MS/MS+and+versatile+peptide+fragmentation+modes.">Ultra high resolution linear ion trap orbitrap mass spectrometer (orbitrap elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes.</a> Molecular &amp; Cellular Proteomics : MCP. 11 (3), 10-1074 (2012).</li><li>Michalski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+proteomics+using+Q+exactive,+a+high-performance+benchtop+quadrupole+orbitrap+mass+spectrometer.">Mass spectrometry-based proteomics using Q exactive, a high-performance benchtop quadrupole orbitrap mass spectrometer.</a> Molecular &amp; Cellular Proteomics : MCP. 10 (9), (2011).</li><li>Snyder, N. W., Khezam, M., Mesaros, C. A., Worth, A., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untargeted+Metabolomics+from+Biological+Sources+Using+Ultraperformance+Liquid+Chromatography-High+Resolution+Mass+Spectrometry.">Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry.</a> UPLC-HRMS). J. Vis. Exp. (75), (2013).</li><li>Gika, H. G., Theodoridis, G. A., Wingate, J. E., Wilson, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-day+reproducibility+of+an+HPLC-MS-based+method+for+metabonomic+analysis:+Application+to+human+urine.">Within-day reproducibility of an HPLC-MS-based method for metabonomic analysis: Application to human urine.</a> Journal of Proteome Research. 6 (8), 3291-3303 (2007).</li><li>Want, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+metabolic+profiling+procedures+for+urine+using+UPLC-MS.">Global metabolic profiling procedures for urine using UPLC-MS.</a> Nature Protocols. 5 (6), 1005-1018 (2010).</li></ol>

The most critical steps for successful metabolite profiling in cells using this protocol are: 1) controlling the growth medium and careful extraction of the cells; 2) adjusting the LC method based on MS method setup to ensure there are enough (usually at least 10) data points across a peak for quantitation; 3) doing a low mass calibration before running samples; 4) injecting no more than 5 ml to avoid retention time shifting and peak broadening caused by water; and 5) preparing and running samples for comparison in the s...

Metabolomics, defined as an experiment that measures multiple metabolites simultaneously, has been an area of intense interest.&#160; Metabolomics provides a direct readout of molecular physiology and has provided insights into development and disease such as cancer1-4.&#160; Nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS) are among the most commonly used instruments5-9. &#160;NMR, especially has been used for flux experiments since heavy isotope labeled compounds, such as 13C labeled metabolites, are NMR-active10,11. &#160;However, this strategy requires relatively high sample purity and large sample quantity, which limits its applications in metabolomics. &#160;Meanwhile, data collected from NMR needs intensive analysis and compound assignment of complex NMR spectra is difficult.&#160;GC-MS has been widely used for polar metabolites and lipid studies, but it requires volatile compounds and therefore often derivatization of metabolites, which sometimes involves complex chemistry that can be time consuming and introduces experimental noise.
Liquid chromatography (LC) coupled to triple quadrupole mass spectrometry uses the first quadrupole for selecting the intact parent ions, which are then fragmented in the second quadrupole, while the third quadrupole is used to select characteristic fragments or daughter ions. &#160;This method, which records the transition from parent ions to specific daughter ions, is termed multiple reaction monitoring (MRM). &#160;MRM is a very sensitive, specific, and robust method for both small molecule and protein quantitation12-15,21.&#160; However, MRM does have its limitations. To achieve high specificity a MRM method needs to be built for each metabolite. This method consists of identifying a specific fragment and corresponding optimized collision energy, which requires pre-knowledge of the properties of the metabolites of interest, such as chemical structure information. Therefore, with some exceptions involving the neutral loss of common fragments, it is not possible to identify unknown metabolites with this method.&#160;

In the recent years, high-resolution mass spectrometry (HRMS) instruments have been released, such as the LTQ-orbitrap and Exactive series, the QuanTof, and TripleTOF 560016-18,22. HRMS can provide a mass to charge ratio (m/z) of intact ions within an error of a few ppm. Therefore, an HRMS instrument operated by detecting all precursor ions (i.e. full scan mode) can obtain direct structural information from the exact mass and the resulting elemental composition of the analyte, and this information can be used to identify potential metabolites.&#160; Indeed, all information about a compound can be obtained with an exact mass, up to the level of structural isomers. &#160;Also, a full scan method does not require previous knowledge of metabolites and does not require method optimization. &#160;Moreover, since all ions with m/z falling into the scan range can be analyzed, HRMS has a nearly unlimited capacity in terms of the number of metabolites that can be quantified in a single run compared to the MRM method. HRMS is also comparable to a triple quadrupole MRM in quantitative capacity due to the short duty cycle resulting in a comparable number of data points that can be obtained in a full MS scan. Therefore, HRMS provides an alternative approach for quantitative metabolomics. Recently, an improved version of HRMS termed Q-Exactive mass spectrometry (QE-MS) can be operated under the switching between positive and negative modes with sufficiently fast cycle times in a single method, which expands the detection range19.&#160; Here we describe our metabolomics strategy using the QE-MS.&#160; &#160;

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Positive calibration mix</td>
			<td style="width:117px;">Thermo Scientific</td>
			<td style="width:120px;">#88323</td>
			<td style="width:207px;">It is light sensitive. Store at 4 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Negative calibration mix</td>
			<td>Thermo Scientific</td>
			<td>#88324</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Diazinon</td>
			<td style="width:117px;">Sant Cruz Biotechnology</td>
			<td>#C0413</td>
			<td style="width:207px;">It causes eyes irritation, so work in hood. Store at 4 &#176;C</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">H-ESI needle insert</td>
			<td>Fisher Scientific</td>
			<td>#1303200</td>
			<td style="width:207px;">This could be replaced or cleaned with 5 % Formic acid/water (remove rubber ring) if clogged.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xbridge amide column</td>
			<td>Waters</td>
			<td>#186004860</td>
			<td style="width:207px;">Guard column is recommend to increase column lifetime.</td>
		</tr>
	</tbody>
</table>

a strategy for sensitive, large scale quantitative metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

The accuracy of metabolomics data highly depends on the LC-QE-MS instrument performance. To assess whether the instrument is operating in good condition, and whether the method applied is proper, several known metabolite LC peaks are extracted from the total ion chromatography (TIC), as shown in Figure 1. Polar metabolites, including amino acids, glycolysis intermediates, TCA intermediates, nucleotides, vitamins, ATP, NADP+ and so on have good retention on the column and good peak shapes in th...

Watch this Scientific Journal Video about A Strategy for Sensitive, Large Scale Quantitative Metabolomics at JoVE.com

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

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Research

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Biology

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.

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

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

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.

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

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...