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Representative Results






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

Published: August 24th, 2018



15th Medical Department, Medical Faculty Mannheim, Heidelberg University, 2Department of Internal Medicine, Heidelberg University, 3German Center for Diabetes Research (DZD), 4European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University
* These authors contributed equally

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.

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.

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....

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NOTE: This protocol is divided into five sections. In sections 1–3, 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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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.......

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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.......

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


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

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