This study was supported by the Deutsche Forschungsgemeinschaft (DFG) within the IRTG 1874 &#34;Diabetic microvascular complications&#34; and CRC 1118 &#34;Reactive metabolites as a cause for diabetic late complications&#34;. C. elegans strains N2 and CL2166 were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

NOTE: This protocol is divided into five sections. In sections 1&#8211;3, the main protocol to culture C. elegans at a large-scale is presented. Sections 4 and 5 provide additional protocols for the assessment of exemplified metabolites occurring in diabetic metabolites. In detail, section 1 describes a general large-scale culture on plates. Section 2 focuses on the transfer of large amounts of C. elegans, whereas section 3 explains the harvesting of a large-scale sample. Section 4 explains the protein ...

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H., 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=Retinal+neurodegeneration+may+precede+microvascular+changes+characteristic+of+diabetic+retinopathy+in+diabetes+mellitus.">Retinal neurodegeneration may precede microvascular changes characteristic of diabetic retinopathy in diabetes mellitus.</a> Proceedings of the National Academy of Sciences. 113 (19), E2655-E2664 (2016).</li><li>Chilelli, N. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+methylglyoxal+by+stable+isotopic+dilution+analysis+LC-MS/MS+with+corroborative+prediction+in+physiological+samples.">Measurement of methylglyoxal by stable isotopic dilution analysis LC-MS/MS with corroborative prediction in physiological samples.</a> Nature Protocols. 9 (8), 1969-1979 (2014).</li><li>Bradford, M. 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+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 7 (72), 248-254 (1976).</li><li>Ahmed, N., Argirov, O. K., Minhas, H. S., Cordeiro, C. A., Thornalley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+advanced+glycation+endproducts+(AGEs):+surveying+AGEs+by+chromatographic+assay+with+derivatization+by+6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate+and+application+to+Nepsilon-carboxymethyl-lysine-+and+Nepsilon-(1-carboxyethyl)lysine-modified+albumin.">Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to Nepsilon-carboxymethyl-lysine- and Nepsilon-(1-carboxyethyl)lysine-modified albumin.</a> Biochemical Journal. 364 (Pt 1), 1-14 (2002).</li><li>Thornalley, P. 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=Quantitative+screening+of+advanced+glycation+endproducts+in+cellular+and+extracellular+proteins+by+tandem+mass+spectrometry.">Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry.</a> Biochemical Journal. 375 (Pt 3), 581-592 (2003).</li><li>Karachalias, N., Babaei-Jadidi, R., Ahmed, N., Thornalley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+fructosyllysine+and+advanced+glycation+end+products+in+the+kidney,+retina+and+peripheral+nerve+of+streptozotocin-induced+diabetic+rats.">Accumulation of fructosyllysine and advanced glycation end products in the kidney, retina and peripheral nerve of streptozotocin-induced diabetic rats.</a> Biochemical Society Transactions. 31, 1423-1425 (2003).</li><li>Morcos, 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=Glyoxalase-1+prevents+mitochondrial+protein+modification+and+enhances+lifespan+in+Caenorhabditis+elegans.">Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans.</a> Aging Cell. 7 (2), 260-269 (2008).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> Journal of Visualized Experiments. (64), e4019 (2012).</li><li>Lagido, C., McLaggan, D., Glover, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Screenable+In+Vivo+Assay+for+Mitochondrial+Modulators+Using+Transgenic+Bioluminescent+Caenorhabditis+elegans.">A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans.</a> Journal of Visualized Experiments. (105), e53083 (2015).</li><li>Takamiya, S., Mita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+purification+of+active+liquid-cultured+Caenorhabditis+elegans+using+a+modified+Baermann+apparatus.">Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus.</a> Parasitology International. 65 (5 Pt B), 580-583 (2016).</li><li>Cornaglia, M., Lehnert, T., Gijs, M. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+systems+for+high-throughput+and+high-content+screening+using+the+nematode+Caenorhabditis+elegans.">Microfluidic systems for high-throughput and high-content screening using the nematode Caenorhabditis elegans.</a> Lab Chip. 17 (22), 3736-3759 (2017).</li><li>Zhu, G., Yin, F., Wang, L., Wei, W., Jiang, L., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+type+2+diabetes-like+hyperglycemia+in+C.+elegans+on+a+microdevice.">Modeling type 2 diabetes-like hyperglycemia in C. elegans on a microdevice.</a> Integrative Biology. 8 (1), 30-38 (2016).</li></ol>

This protocol presents a reliable approach for the large-scale culturing of C. elegans to obtain quantitative results. Findings from the literature could be replicated as shown in the Representative Results. Even though this protocol for the collection of large-scale samples of C. elegans seems like a straight-forward method, there are certain pitfalls to take into account. Regarding the synchronization of the nematode population, this protocol describes an approach by bleaching the pop...

Protein analyses, such as a western blot or mass spectrometry, require milligrams of protein. This yield requires a large-scale culturing of hundreds of C. elegans, which can be accomplished either by liquid culture or on solid media transferring the nematodes by washing. Fluid and shear stress induces the expression of epithelial sodium channels (ENaC), which could increase the osmotic stress through an increased uptake of sodium, potentially altering the lifespan of C. elegans and affecting metabolic analyses1. Therefore, some critical steps in this protocol for the plate-based approach take the reduction of stress affecting experimental variability into account. Liquid culture, on the other hand, influences the phenotype of the nematodes and complicates the culture and collection of an exact number of nematodes2. Moreover, reactive substances can be altered by media components and may distribute unevenly before reaching the nematodes. Regarding the limitations of liquid culture, this protocol provides an alternative approach to culturing large-scale samples of C. elegans.
C. elegans is a model organism with a distinct network of 302 neuronal cells, making up one-third of all its cells3. Since its introduction into science, many homologous and orthologous genes have been described, amplifying its value as a model for medical research. Recently, evidence for neurological impairment in diabetic retinopathy, preceding vascular damage, has been presented4. C. elegans is lacking blood vessels, but contains a distinct neuronal network, making it a suitable model to investigate neuronal alterations apart from vascular ones. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. Biochemical alterations in diabetic complications involve the formation of AGEs, which further influence the formation of ROS in response to hyperglycemia5. AGEs are found in C. elegans and contribute to neuronal damage6. Chronic diseases are often caused by complex, polygenic processes requiring a multiparametric approach for the assessment of their underlying mechanisms, as exemplified here with the assessment of diabetic complications. This protocol can be of use for obtaining multiple parameters simultaneously, as well as subsequently. Increased comparability and reproducibility of a multiparametric approach can be achieved by omitting the morphological and metabolic differences between liquid and solid media culture.

<table>
	<tbody>
		<tr>
			<td>E. coli OP50</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans N2</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans CL2166</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 60 x 15 mm</td>
			<td>Greiner One</td>
			<td>628161</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric pipet, glas, 10 mL</td>
			<td>Neolab</td>
			<td>E-0413</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>04693124001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-denaturing lysate buffer:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl, pH 8</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodiumchloride (NaCl)</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E5391</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates, transparent bottom</td>
			<td>Brand</td>
			<td>781611</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite M200, plate reader</td>
			<td>Tecan</td>
			<td>30017581</td>
			<td></td>
		</tr>
		<tr>
			<td>Zirconium Oxide Beads, 0.5 mm</td>
			<td>Next advance</td>
			<td>ZROB05-RNA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender, homogenizer</td>
			<td>Next advance</td>
			<td>BBX24</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin from porcine gastric mucosa</td>
			<td>Sigma</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymol</td>
			<td>Sigma</td>
			<td>T0501</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E/ Protease from Streptomyces griseus </td>
			<td>Sigma</td>
			<td>P6911</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma</td>
			<td>P43339</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolidase from Porcine Kidney</td>
			<td>Sigma</td>
			<td>P6675</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminopeptidase from Aeromonas proteolytica</td>
			<td>Sigma</td>
			<td>A8200</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>Merckmillipore</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Materials for plate culture are described in Reference 6.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

plate-based large-scale cultivation of caenorhabditis elegans: sample preparation for the study of metabolic alterations in diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Here examples of creating a large-scale C. elegans culture for applications in diabetes research are presented. It can be of interest to relate the parameters to a single animal, rather than to normalize it to the total protein concentration. In an assay requiring a small number of nematodes, this can be easily accomplished by counting the nematodes. For a large-scale C. elegans culture involving hundreds of nematodes per experimental group, this approach is inconvenient...

Watch this Scientific Journal Video about Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes at JoVE.com

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

In the C.elegans field, this method can help answer key questions which require the production of large nematode populations and samples using plate culture. The main advantages of this protocol are its easy establishment, and its ease to description, including pitfalls and applications. After obtaining a synchronized population of nematodes, add approximately one milliliter of M nine buffer to the plates, and carefully distribute the buffer by swinging the plates.After this, collect the nematodes in a 15 milliliter tube. Now, invert the tube and use a microscope to evaluate the density of the nematodes. Adjust the density of the suspension as needed, and continue to evaluate it under the microscope.Apply 150 microliters of the suspension directly to the plates with modified pipet tips. To ensure an equal distribution of nematodes between the plates, invert the tube of suspension at regular intervals. First, wash the nematodes using a modified pipet tip with approximately 500 microliters of M nine buffer.Using the same buffer, continue detaching the nematodes until most of the animals are collected in suspension. Next, take up the nematodes with a modified pipet tip, and transfer them to a plate with OP50. The modified pipet tips decrease shear stress and damage to the nematodes because of a larger diameter.Then, allow the plate to dry. Before collecting samples, prepare liquid nitrogen to snap freeze the collected nematodes. Then, place M nine buffer on ice.Apply one milliliter of M nine buffer to each plate, and gently swirl to wash each plate. Then, take up the volume of M nine buffer, and transfer it to new 15 milliliter tubes. Centrifuge the tube briefly at 800 times g, before removing most of the M nine buffer.Next, wash the samples three times with 10 milliliters of M nine buffer to remove any bacteria. Using a glass pipette, transfer each pellet to an empty 1.5 milliliter tube. Use the glass pipette to take up one milliliter of M nine buffer from the second 1.5 milliliter tube, to rinse the nematodes from the pipette wall.Then, wash the remaining nematodes from the 15 milliliter tubes, and transfer them to the sample tubes. Next, centrifuge the sample tubes at 19, 000 times g for one minute, and remove as much supernatant as possible. Then, seal each tube with paraffin film, and snap freeze them in liquid nitrogen immediately.Store the tubes at minus 80 degrees Celsius until lysate preparation. Add the same volumes of 0.5 millimeter zirconium oxide beads, and twice the volume of lysate buffer with proteinase inhibitor cocktail to each frozen sample. Then, place the tubes in a homogenizer for one minute on speed nine.Centrifuge the samples for 30 minutes at four degrees Celsius, and 19, 000 times g. Finally, transfer the supernatant to a new tube on ice, and store it at minus 80 degrees Celsius for future experiments. In this protocol, large quantities of C.elegans were cultured for applications in diabetes research.The number of nematodes collected correlated to the protein yield of the lysate, demonstrating that reproducible quantitative protein isolation can be achieved using this protocol. Mass spectrometric analyses of the reactive metabolite methylglyoxal, and advanced glycation end products formed by methylglyoxal exposure are shown here. Glucose-treated nematodes and controls were compared.Transferring nematodes via pipette can create shear stress, potentially harming the nematodes. Oxidative stress levels in pipetted nematodes and nematodes transferred with a metal wire did not differ using this protocol. Following this procedure, other methods like western blot can be performed, in order to answer additional questions in proteomics.This protocol also facilitates experiments that relate multiple parameters to the same population.

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Biochemistry

6.2K visualizações.  Heidelberg University. In the C.elegans field, this method can help answer key questions which require the production of large nematode populations and samples using plate culture. The main advantages of this protocol are its easy establishment, and its ease to description, including pitfalls and applications. After obtaining a synchronized population of nematodes, add approximately one milliliter of M nine buffer to the plates, and carefully distribute the buffer by swinging the plates.After this, collect t...