We thank Silke Classen from the Research Institute for Ecosystem Analysis and Assessment (gaiac), RWTH Aachen for her assistance in the establishment of the group-specific primer sets used in this protocol. We also thank Peter Martin and Laura Schynawa from the University of Kiel for the cooperation in the water mites&#39; experiments. We also thank the technical lab assistants for keeping the lab running smoothly and preparing the register of companies and catalogue numbers. This work was supported by the German Research Foundation (DFG; project GE 2219/3-1).

<ol>
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The authors have nothing to disclose.

Here, we present an easy and cost-efficient method for PCR-based determination of predator-prey interactions from field samples via group-specific primers for rDNA regions, which can be combined with other non-molecular analyses of the same specimens. However, there are several critical steps within the protocol. Firstly, assuring the specificity of the primers used is essential. Secondly, it is crucial to avoid contamination and loss of gut content material during the preparation of the gastro-intestinal tract and the subsequent extraction of DNA. While cross-sample contamination can lead to false positive detection of prey DNA, loss of gut content material might lead to missing rarely consumed prey species. Verification of the presence of rDNA belonging to the respective subunit and the functional efficiency of templates used for the analyses (section 2 of the protocol) is essential for gathering representative information about the prey consumed. This is in particular crucial, if no DNA of prey groups the consumer is supposed to feed on can be detected25,27. Furthermore, when preparing the PCRs (steps 3.1, 5.1.1 and 5.1.2) it is crucial that the mixture and protocol used exactly fit the criteria in which the respective primer was specified to a certain group of taxa. Otherwise, either amplification of DNA within the PCR reaction might fail completely, or the DNA of taxa not targeted might be amplified as well. Therefore, it is obligatory to use positive and negative controls within each PCR run to assure that the PCR reaction worked and no contamination occurred. Additionally, it is necessary that the array of the samples for each PCR run is documented exactly to enable the correct assignment of prey consumed to the respective consumer specimen. For this task, particular caution needs to be taken while pooling PCR products for parallel detection of multiple prey groups via automated fragment analyses (step 5.2.3).
For the detection of amplified fragments using agarose gel electrophoresis (steps 3.2 and 3.3) agarose gels should be as thin as possible and care has to be taken during the visual inspection of the image to avoid missing fragments with low visual resolution. For results and an appropriate conclusion about prey consumption by a consumer, it is crucial that even rarely consumed prey taxa are being recognized. Thick agarose gels diminish the visibility of potentially low amounts of prey DNA and thus reduce the probability to miss rarely consumed prey and introduce uncertainties in the conclusions drawn from the analysis. Furthermore, from a health perspective, using a less- or non-toxic alternative than ethidium bromide for staining the gels might be advisable. The protocol for subsequent further downstream analysis of amplified fragments via SSCP analyses using polyacrylamide gels includes some steps that should be performed with particular caution. It is important that gel cassette assembled is leak-proof (step 4.1.3) and the polyacrylamide solution is given enough time to polymerize completely (step 4.1.6). Opening the cassette too early will lead to leaking liquid acrylamide solution and, despite the fact that the preparation has to be started from the beginning, extensive cleaning up needs to be done as well. Furthermore, transferring the polyacrylamide gel into the staining bath (step 4.7) and from there onto the UV table (step 4.8) needs to be done very carefully due to the instability of such gels. Otherwise, the gel might tear and fragments could be disrupted, which leads to the necessity to repeat the procedure.
One major requirement for successfully processing data received from automated fragment analyses is that the sample&#39;s internal size standard closely matches the size standard pattern given by the manufacturer (step 5.3.3) to enable determination of the appropriate fragment length. This is especially crucial because otherwise, different fragments, labelled with the same fluorescent staining dye, might be assigned to the wrong prey taxa and therefore lead to a complete misjudgment of predator-prey interactions. Furthermore, electropherograms often show background noise. To establish confidence in the interpretation, it is essential that the peaks of the internal size standard are significantly higher than the background noise of the sample. Consequently, peaks for each marker/primer should only be counted if they are significantly higher than the background noise.
This protocol can be modified to detect several groups of prey taxa of interest within various consumers of interest. Admittedly, group-specific primers may not already be available for every potential prey taxon of interest. Nevertheless, group-specific primers are available not only for several freshwater macroinvertebrate taxa28, but also for a broad range of other taxa (e.g., several marine macroinvertebrate taxa16,30 and common taxa of flying insects31). Thereby, the selection of group-specific primer sets suitable to amplify DNA of all the prey species of a given taxon, but unsuccessful in the amplification of DNA from species not belonging to this taxon, is essential32. Many group-specific primers that are now available have been specified for a given range of taxa (e.g., 130 taxa of freshwater macroinvertebrates common in the River Rhine system28). Therefore, to avoid false-negative or false-positive results, the specificity of such primers should be rechecked for the range of taxa common in the ecosystem of interest prior to their application for target taxa and consumers not included in the range of taxa for which the primers had been established. Furthermore, if those molecular analyses are not to be combined with other analyses of the exact same individual, dissection of the gastrointestinal tract might be skipped.
Nevertheless, dissecting the gastrointestinal tract of larger specimens also prevents failure of DNA extraction because of too much material, and reduces the presence of consumer-DNA in the sample extract. However, studies have shown that using specific blocking primers, which attach to the DNA of the consumer and thereby block it, for the PCR reaction can effectively enhance the amplification of rare sequences in mixed samples33. Therefore, dissection of the gastrointestinal tract can be avoided, even when dealing with larger consumer specimens, by the use of specific blocking primers. For parallel detection of amplicons derived from multiple group-specific primer sets via automated fragment analyses, care should be taken in the choice of staining dye as label. Thereby, primer sets amplifying fragments of approximately the same length should be labelled with clearly distinguishable fluorescent dyes. Moreover, there are a range of capillary electrophoresis systems available from several manufacturers. Some of these systems supposedly allow the determination of multiple fragments even via unspecific dyes. The protocol presented here could easily be adjusted to detect amplified fragments with any of those systems. Apart from that, to reduce the time needed for the analyses, it would be possible to optimize single multiplex-PCR assays to amplify DNA of prey groups of one fragment analysis batch simultaneously (for instance, simultaneous amplification of 12 prey organisms of beetles34 or up to six flying insect taxa31).
A potential disadvantage of gut content analyses in general, and therefore also the analyses described in this protocol, is the low temporal resolution. With such methods, it is only possible to identify recently ingested organisms, which might lead to uncertainties in the importance of single prey organisms2. However, we demonstrated that genetic analysis according to this protocol can easily be combined with analyses of stable isotopes (&#948;15N and &#948;13C) of the exact same consumer specimens25,27. As a time-integrating natural tracer of predator-prey interactions, stable isotope analyses are frequently used to characterise food web structures1, but potential overlapping isotopic values from different prey species often impede an exact definition of predator-prey interactions2. Therefore, using this protocol for genetic analyses in combination with stable isotope analyses to overcome the disadvantages of each method can further our understanding of food webs and their relationship to nutrient or energy fluxes.

Predator-prey interactions, which constitute the majority of trophic interactions and food web dynamics, are key aspects to characterize the fluxes of matter and energy throughout food webs within and between ecosystems, which is one of the major goals of ecology1. The determination of the source and flow of carbon and nutrients is furthering the understanding of ecological connectivity between ecosystems2. However, ecosystems, such as rivers and their catchments, are not only linked by fluxes of organic matter and nutrients but also by the movements of organisms3. Thus, habitat alterations interrupting the flow of resources that link those systems can strongly alter the food webs of both ecosystems, not only directly but also indirectly by changing the respective composition of predator-prey communities. For instance, changes of food webs have been shown to be linked to the movements of single predator species (e.g., rainbow trout)4. Such changes potentially threaten biodiversity and the functioning of aquatic ecosystems. Therefore, analyzing predator-prey interactions in the field is essential to determine the impact of human-induced environmental changes, such as water management practices, on the native biodiversity of aquatic ecosystems.
Since tracking trophic linkages is difficult in complex systems, several approaches have been established that enable the assessment of feeding interactions in the field5. Traditionally, investigations of feeding interactions in the field are based on visual identification of prey remains in dissected guts and require an extensive knowledge about morphologic prey diversity. Visual gut content analyses have provided insights in resource use of several groups of consumers (e.g., seasonal variation in diet of lobster6 and fish7,8 or feeding preferences of copepods). However, the physical process of digestion makes visual gut content analyses difficult and usually misses soft bodied prey-organisms9. For species feeding by liquid ingestion or for some invertebrate consumers that intensively comminute their food before ingestion, like amphipods, visual identification of prey species in gut contents is impossible10. Due to these limitations, molecular analysis provides a promising alternative.
Molecular analyses have now become a common tool allowing rapid and precise prey detection in gut contents. The range of such techniques is diverse: strategies based on monoclonal antibodies or polymerase chain reactions (PCR) are often used, because of their high specificity and sensitivity11. The development of new monoclonal antibodies is time- and cost-intensive, therefore, the application of other molecular techniques is more useful when antibodies do not already exist11. Another common approach is the amplification of regions of deoxyribonucleic acid (DNA), like ribosomal ribonucleic acid (RNA) genes present in most species, using universal primer sets12,13. When using this technique, it is often not possible to identify the whole range of prey organisms within mixed sources of DNA14. An effective approach to avoid such a drawback is to use group-specific primer sets for genetic gut content analyses. Designed to amplify only DNA regions of particular target groups and exclude all other species15,16, group-specific primers enable identification of prey organisms on the taxonomic level of the specified groups without time- and cost-intensive secondary analyses. However, like all gut content analysis, such analyses provide only a snapshot of feeding behavior. Therefore, combining molecular gut content analyses with analyses of time-integrating natural tracers (e.g., stable isotopes) is considered beneficial1,2.
Here, we describe a detailed method for PCR-based investigations of predator-prey interactions using group-specific primer sets for nuclear ribosomal DNA (rDNA) regions to be combined with stable isotope analyses of the same specimen. We describe the detection of the DNA of single prey groups via agarose gel electrophoresis. Additionally, we present an opportunity for further downstream analyses of PCR products of such group-specific primers applicable whenever a higher taxonomic resolution than the primers&#39; specificity is required. Because single stranded DNA (ssDNA) fragments form tertiary conformations that are determined by their primary sequence17, small variations in fragments amplified by such group-specific primers lead to conformational changes. Such changes can be detected by single strand conformation polymorphism (SSCP) analyses with polyacrylamide gels17,18, enabling a more precise identification of prey organisms (down to the species level).
While agarose gel electrophoresis is a common and inexpensive tool to visualize DNA fragments and determine their approximate length19, the resolution depends on the amount of DNA and the staining dye used20. Usually, the visualization is straight forward when working with pure DNA samples, but potentially low amounts of prey DNA in the gut contents of consumers can complicate the scoring of agarose gel electrophoresis results. Still, this detection method is feasible to screen a low number of consumer specimens from the field for one or a few prey groups, but complication in the scoring makes the screening of a high number of samples for multiple prey taxa extremely time intensive and thus impracticable. A more sensitive detection method is the automated analysis of fragments via capillary electrophoresis, which additionally allows the determination of the exact length of fragments21. Several microsatellite based studies have shown that by using different fluorescent dyes as labels, it is possible to detect and determine different fragments of comparable length by automated fragment analysis22,23,24. Therefore, we also present a detailed protocol for parallel detection of DNA from multiple prey groups using PCR with labeled group-specific primer sets and detection via automated fragment analysis with an automated sequencer. Additionally, we present results from a case study demonstrating that the detection of prey DNA via automated fragment analysis is an approach which also enables a relative quantification of ingested prey.

<table>
	<tbody>
		<tr>
			<td>liquid nitrogen</td>
			<td>Any</td>
			<td>NA</td>
			<td>For transport of field samples of macroinvertebrates to the laboratory.</td>
		</tr>
		<tr>
			<td>1.5 ml reaction tubes - Safelock</td>
			<td>Any</td>
			<td>NA</td>
			<td>For collection of consumer specimens in the field and subsequent conservation in liquid nitrogen reaction tubes do not necessarily need to be PCR clean. To avoid exploding of the tubes, the lid of the tubes should be punctured with a needle.</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Any</td>
			<td>NA</td>
			<td>Make sure magnification is suitable to prepare the gastro-intestinaltract of the size of consumer of interest.</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Any</td>
			<td>NA</td>
			<td>To dissect the gastro-intestinal tract of consumer specimens under the stereomikroscope.</td>
		</tr>
		<tr>
			<td>Stainless steel foceps: Dumont forceps No. 7</td>
			<td>Bioform</td>
			<td>B40d</td>
			<td>This curved stainless steel forceps can sometimes be very helful to hold specimen fixed inbetween the curved part without puncturing the gastro-intestinal tract.</td>
		</tr>
		<tr>
			<td>Stainless steel foceps: Dumont forceps Electronic No. 5</td>
			<td>Bioform</td>
			<td>B40b</td>
			<td>This fine stainless steel forceps is ideally suited for the preparation of the gastro-intestinal tract of consumer specimens.</td>
		</tr>
		<tr>
			<td>DNA-ExitusPlus</td>
			<td>AppliChem</td>
			<td>A7409,1000RF</td>
			<td>Decontamination solution for the removal of DNA and RNA contaminations</td>
		</tr>
		<tr>
			<td>fuzz-free paper towel</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab scale</td>
			<td>Any</td>
			<td>NA</td>
			<td>Precision needs to be at least 0.01 g.</td>
		</tr>
		<tr>
			<td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &#956;L)</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 2.0 ml</td>
			<td>VWR</td>
			<td>211-2165</td>
			<td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 1.5 ml</td>
			<td>VWR</td>
			<td>211-2164</td>
			<td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td>
		</tr>
		<tr>
			<td>Sodium chloride &#8805;99.5%, p.a., ACS, ISO</td>
			<td>carlroth</td>
			<td>3957.3</td>
			<td>For salt extraction buffer (SEB) and 5M NaCl-solution required for extraction of DNA.</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)-aminomethane Tris</td>
			<td>carlroth</td>
			<td>4855.2</td>
			<td>For salt extraction buffer (SEB) and TBE buffer</td>
		</tr>
		<tr>
			<td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &#956;L)</td>
			<td>carlroth</td>
			<td>8043.1</td>
			<td>For salt extraction buffer (SEB) and TBE buffer</td>
		</tr>
		<tr>
			<td>Boric acid &#8805;99.8 %, p.a., ACS, ISO</td>
			<td>carlroth</td>
			<td>6943.2</td>
			<td>For TBE buffer. Not needed if commercialy available TBE buffer is obtained.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>carlroth</td>
			<td>4360.1</td>
			<td>For extraction of DNA</td>
		</tr>
		<tr>
			<td>Proteinase K lyophil.</td>
			<td>carlroth</td>
			<td>7528.1</td>
			<td>Prepare a working solution containing 10 mg/ml.</td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Quiagen</td>
			<td>85300</td>
			<td>Bead mill for homogenization of the samples.</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads, 5 mm</td>
			<td>Quiagen</td>
			<td>69989</td>
			<td>For homogenization of samples.</td>
		</tr>
		<tr>
			<td>Heating Thermoshaker</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge: himac CT 15 RE</td>
			<td>Hitachi</td>
			<td>NA</td>
			<td>Refrigerated centrifuge used for centrifugation during the extraction of DNA (Protocol 1).</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>carlroth</td>
			<td>6752.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99.8 %, p.a.</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Molecular biology grade (ddH2O)</td>
			<td>AppliChem</td>
			<td>A7398,1000</td>
			<td>nuclease-free (stream sterilized and DEPC treated) water used for re-suspension of DNA and whenever refered to ddH2O within the manuscript</td>
		</tr>
		<tr>
			<td>Unmodified Olegonucleotides</td>
			<td>Eurofins MWG Operon</td>
			<td>NA</td>
			<td>Primers unlabeled</td>
		</tr>
		<tr>
			<td>Modified olegonucleotides</td>
			<td>Metabion International AG</td>
			<td>NA</td>
			<td>Primers labelled</td>
		</tr>
		<tr>
			<td>peqGOLD Taq all inclusive</td>
			<td>VWR Peqlab</td>
			<td>01-1000</td>
			<td>Taq all inclusive for PCR reaction mixtures (reaction buffer Y and S, dNTPs and MgCl2)</td>
		</tr>
		<tr>
			<td>Primus 96 advanced gradient thermocycler or peqStar96x Universal Gradient</td>
			<td>Peqlab</td>
			<td>NA</td>
			<td>Primer sets were originally established on a Primus 96 advanced gradient thermocycler. As this cycler was quite old, we than checked if our primer sets are efficient and specific under the same conditions using the peqSta96x (which also could simulate the older model). However, tests showed that our primers work well and are still specific (see Koester et al. 2013) without using the simulation of the old machine.</td>
		</tr>
		<tr>
			<td>Standard 96 Well PCR Plates</td>
			<td>VWR Peqlab</td>
			<td>732-2879</td>
			<td>Used for PCR reactions.</td>
		</tr>
		<tr>
			<td>Low Profile Tube Stripes 0.15 ml with Flat Cap Stripes</td>
			<td>VWR Peqlab</td>
			<td>732-3223</td>
			<td>Cap stripes used to close the PCR plates.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Peqlab</td>
			<td>35-1020</td>
			<td>Used for gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical flask</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring cylinder</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal electrophoresis unit and respective combs with teeth</td>
			<td>Any</td>
			<td>NA</td>
			<td>For agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply PS3002</td>
			<td>Life Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 1 %</td>
			<td>carlroth</td>
			<td>2218.1</td>
			<td>Used to prepare staining bath for agarose gels.</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>carlroth</td>
			<td>6749.1</td>
			<td>Needded for the denaturation buffer for SSCP analyses.</td>
		</tr>
		<tr>
			<td>Bromphenol blue</td>
			<td>AppliChem</td>
			<td>A3640.0010</td>
			<td>Needed for the loading dye for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>carlroth</td>
			<td>4621.1</td>
			<td>Needed for the loading dye for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>100 bp-DNA-Ladder extended</td>
			<td>carlroth</td>
			<td>T835.1</td>
			<td>As size standard for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>UV-Documentation system</td>
			<td>Any</td>
			<td>NA</td>
			<td>To visualize gel electrophoresis results.</td>
		</tr>
		<tr>
			<td>Rotiphorese NF-Acrylamide/Bis-solution 40 % (29:1)</td>
			<td>carlroth</td>
			<td>A121.1</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Ammonium peroxydisulphate</td>
			<td>carlroth</td>
			<td>9592.2</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>foldback clips (32 mm)</td>
			<td>Any</td>
			<td>NA</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>square-shaped plastic box (21 &#215; 6.5 &#215; 5 cm)</td>
			<td>Any</td>
			<td>NA</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>carlroth</td>
			<td>23.67.1</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>RC 10 Digital chiller</td>
			<td>VWR</td>
			<td>462-0138</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Vertical electrophoresis unit P10DS (OWL Separation Systems)</td>
			<td>VWR</td>
			<td>700-2361</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Notched glass plate</td>
			<td>VWR</td>
			<td>730-0261</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Blank glass plate</td>
			<td>VWR</td>
			<td>730-0260</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Spacers</td>
			<td>VWR</td>
			<td>730-0262</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Comb with 25 teeth</td>
			<td>VWR</td>
			<td>730-0294</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Shaking platform</td>
			<td>Any</td>
			<td>NA</td>
			<td>For constant shaking of the staining bath.</td>
		</tr>
		<tr>
			<td>GelStar Nucleic Acid Stain (Lonza Rockland, Inc.)</td>
			<td>Biozym</td>
			<td>850535 (50535)</td>
			<td>For staining of polyacrylamide gels.</td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit 400bp</td>
			<td>AB Sciex</td>
			<td>608098</td>
			<td>For automated fragment analyses. Size stanard for Batch B.</td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit 600bp</td>
			<td>AB Sciex</td>
			<td>608095</td>
			<td>For automated fragment analyses. Size stanard for Batch A.</td>
		</tr>
		<tr>
			<td>Sample Loading Solution (SLS)</td>
			<td>AB Sciex</td>
			<td>608082</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Sequenzing plate - 96 Well Polypropylene PCR Microplate</td>
			<td>Costar</td>
			<td>6551</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Plate centrifuge, PerfectSpin P</td>
			<td>VWR Peqlab</td>
			<td>NA</td>
			<td>mini plate centrifuge used to spin down PCR products.</td>
		</tr>
		<tr>
			<td>Buffer plate - 96 Well EIA/RIA Clear Flat Bottom Polystyrene Not Treated Microplate</td>
			<td>Corning</td>
			<td>9017</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>GenomeLab Seperation buffer</td>
			<td>AB Sciex</td>
			<td>608012</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Separation Gel - LPAI</td>
			<td>AB Sciex</td>
			<td>608010</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Sequenzer - CEQ8000 Genetic Analysis System</td>
			<td>Beckman Coulter</td>
			<td>NA</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>GeneMarker V1.95</td>
			<td>Softgenetics</td>
			<td>NA</td>
			<td>To analyze results of automated fragment analyses.</td>
		</tr>
	</tbody>
</table>

laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

We successfully used steps described in this protocol for diet analyses within different studies. In this section, we present examples describing the applicability of the different sections of the protocol.
As an example, to demonstrate the effectiveness of group-specific primer sets established by the authors28 and the potential of further downstream analyses of PCR products by SSCP analyses, a feeding experiment was conducted. Individuals of Dikerogammarus villosus as predators and Caenis spp. larvae as prey were captured in the River Rhine and backwaters near Karlsruhe (Germany). In the laboratory, predator and prey taxa were kept separately in filtered stream water at 20 &#176;C and were starved for 36 hours. For the experiment, amphipod specimens were placed in beakers with filtered stream water (100 mL) individually, fed with two to three Caenis larvae for 30 min, and subsequently frozen in liquid nitrogen. The gastrointestinal tract of each D. villosus was dissected and DNA was extracted as described in Protocol 1. DNA extracts were tested with the Caep28S primer set suitable to detect Caenis spp. using the appropriate PCR protocol28 as described in Protocol 3. To verify differences between amplified fragments of different Caenis species, PCR was conducted with the gut content samples and pure DNA samples of three Caenis reference species. Agarose gel electrophoresis led to single bands of double stranded DNA, which cannot be distinguished between the different Caenis species with this type of electrophoresis (Figure 2A). In contrast, by SSCP gel electrophoresis, as described in Protocol 4, it was possible to identify prey organisms to species level by comparing the gut content samples with the reference samples (Figure 2B). The SSCP fragment pattern of the three reference taxa Caenis beskidensis (Figure 2B, lane 1), Caenis horaria (Figure 2B, lane 2), and Caenis luctuosa&#160;(Figure 2B, lane 3) consisted of four bands with differentiable patterns. Two gut content samples (Figure 2B, lane 4 and 5) had a fragment pattern equal to that of C. luctuosa (Figure 2B, lane 3). The fragment pattern of the third gut content sample (Figure 2B, lane 6) was identical to that of C. horaria (Figure 2B, lane 2). Sequence analyses of the two gut content samples identified as C. luctuosa and C. horaria (Figure 2B, lane 4 and 6) and the respective reference species verified this result (see electronical supplement alignment fasta file).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56132/56132fig2.jpg" /> 
Figure 2: Original image of (A) agarose gel electrophoresis and (B) SSCP analysis of PCR fragments of gut content and reference samples amplified with the Caep28S primer set. Lanes 1-3 show the reference samples of the species Caenis beskidensis (1), Caenis horaria (2) and Caenis luctuosa (3). In lanes 4-6, the fragment pattern of different gut content samples of the amphipod Dikerogammarus villosus are presented. Lane 7 shows the fragment pattern of a 100 bp DNA ladder. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56132/56132fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Furthermore, a laboratory experiment was conducted in which water mites belonging to the species Hygrobates fluviatilis were fed on chironomid larvae to not only determine if DNA of chironomid individuals consumed can be detected in the water mites, but also to test if the amount of DNA detected depends on the elapsed time since consumption of the prey29. After starving the mites for approximately one week, each mite was offered one chironomid larva as food. Each 3-4 water mite individuals were fixed in ethanol (absolute) for genetic analysis using the Chiro18S primer set specific to the dipteran family Chironomidae28 at 1, 2, 3, 7, 9, 24, 32 and 50 h after being fed29. As a degree of consumption by the water mite individuals, the respective prey individuals were classified into the following five feeding categories: 1 = completely sucked out, integument completely emptied (&#62;90%), 2 = almost completely sucked out (&#62;75-90%), 3 = semi sucked out (&#62;50-75%), 4 = only partially sucked out (&#62;5-50%), 5 = no visible change in the prey&#39;s body structure (&#8804;5%)29. DNA was extracted from the water mites as described in Protocol 1 (without steps 1.2.1 and 1.2.2) and the presence of 18S rDNA in the extract and the functional efficiency of the template were verified as described in Protocol 2. Detection of prey DNA was performed according to Protocol 4 using only the Chiro18S primer set with the forward primer labeled with a fluorescent dye (see Table 2) and the DNA Size Standard Kit - 400. To allow comparisons between samples analysed within different runs of an automated sequencer, the ratio between sample peak height and the peak height of the nearest internal standard (i.e., 360 bp in this case) of each sample were calculated and used as a proxy for the amount of DNA detected. Results showed that chironomid DNA was detectable in virtually all individuals of Hygrobates fluviatilis fed on chironomid larvae29. From the shortest interval (1 h after feeding) to the longest period after feeding (50 h) the relative amount of detected prey DNA was significantly reduced (Figure 3A)29. Furthermore, the relation between time after feeding and the prey classification as a proxy for the ingested amount of prey DNA could be confirmed (Figure 3B)29. The reduction of prey DNA detected with increasing time after feeding was most likely completely reflected by the degradation of DNA and digestion of the prey, because egestion of prey DNA is highly implausible due to the lack of an anus in water mites29. These results confirm the suitability of this approach for the quantification of ingested prey.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56132/56132fig3.jpg" /> 
Figure 3: Relationships between the national ln height-ratio (sample-peak heights/standard360 peak height) and (A) time after feeding of mites and (B) feeding category (n = 23). &#34;Experimental and Applied Acarology, First detection of prey DNA in Hygrobates fluviatilis (Hydrachnidia, Acari): a new approach for determining predator-prey relationships in water mites, 67, 376, P. Martin, M. Koester, L. Schynawa, R. Gergs, (&#169; Springer International Publishing Switzerland 2015)&#34; With permission of Springer. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56132/56132fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To investigate the importance of predation by the invasive amphipod D. villosus in the field, intensive stable isotope analyses of &#948;13C and &#948;15N of D. villosus and potential food resources were furthered by molecular detection of recently ingested prey within the gut contents of the exact same individuals. In total, 206 D. villosus individuals from different sites were collected and the gastrointestinal tract of each individual was dissected and DNA was extracted according to Protocol 1. The presence and functional efficiency of the templates used was assured as described in Protocol 2. Recent predation by the D. villosus individuals on multiple macroinvertebrate prey taxa was tested as described in Protocol 4. Overall, only 16% of the D. villosus gut contents tested positive for DNA belonging to any of the 16 targeted macroinvertebrate prey taxa, and therefore supported the results of the stable isotope analyses that indicated a low predacious feeding behavior of the invasive amphipod in the field27. While, within the genetic analyses, DNA of 12 tested prey taxa was found in at least 1 gut content sample, DNA of the 4 other prey taxa was never detected (Figure 4)27.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56132/56132fig4.jpg" /> 
Figure 4: Histogram illustrating the respective number of D. villosus individuals from all ten sites whose gut contents tested positive for macroinvertebrate prey DNA. PCR was carried out with 16 group-specific primer sets as indicated. &#34;Hydrobiologia, Is Dikerogammarus villosus (Crustacea, Gammaridae) a &#39;killer shrimp&#39; in the River Rhine system?, 768, 2016, 310, Koester Meike, Bayer Bastian, Gergs Ren&#233;, (&#169; Springer International Publishing Switzerland 2015)&#34; With permission of Springer. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56132/56132fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File. Sequence_alignement_feeding_trial.fasta <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56132/Sequence_alignement_feeding_trial.fasta" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers at JoVE.com

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

laboratory-protocol-for-genetic-gut-content-analyses-aquatic

Nanyang Technological University

Research

JoVE Journal

Biology

8.8K Views.  University of Koblenz-Landau. Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Video: Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.