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 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.
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 isolation from the sample and section 5 describes the sample preparation for subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses.
1. Large-scale Culture on Plates
2. Transfer of Large Amounts of Caenorhabditis elegans
3. Harvest of a Large Caenorhabditis elegans Sample
4. Protein Isolation for Mass Spectrometry
NOTE: The protein isolation described here can also be used for other assays (e.g., western blot).
5. Sample Preparation for Liquid Chromatography-tandem Mass Spectrometry Measurements
NOTE: Depending on the parameter of interest, the sample preparation will differ. This protocol focuses on methylglyoxal and AGE determination.
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. In Figure 2, the correlation between the amount of C. elegans and the protein content is shown. As demonstrated here, reproducible quantitative protein isolation can be achieved using this protocol.
The Amadori adduct fructosyl-lysine is one of several AGE precursors formed during diabetes in experimental animal models13. Figure 3 displays LC-MS/MS measurements after a glucose treatment in 12-day-old nematodes and age-matched untreated controls from three independent experiments. The nematodes were homogenized with an addition of 200 µL of lysate buffer. An increased formation of fructosyl-lysine after the high-glucose treatment is presented.
Reactive metabolites such as methylglyoxal are fluctuant and modify proteins quickly14. Therefore, the question remains whether those reactive metabolites actually accumulate in the organism or are modified before reaching their target. Figure 4 confirms the accumulation of methylglyoxal in C. elegans after a treatment with the substance.
Oxidative stress is increased during hyperglycemia6, as well as during shear stress. Figure 5 confirms no difference in the formation of oxidative stress between the glucose-treated group, transferred as in section 2 of the protocol, and the non-transferred group. Comparison of both glucose-treated groups to the untreated control group shows a significant increase in oxidative stress induced by glucose. In contrast, no significant difference between the two glucose-treated groups was observed. These results illustrate that handling nematodes using this protocol does not significantly induce the formation of oxidative stress, which could serve as a confounder in investigations of aging or metabolism.Moreover, findings in the current literature were reproduced using this protocol.
Figure 1: Adequate density of nematodes for a further distribution on plates. (A) This panel shows a macroscopic view. (B) This panel shows a microscopic view, at 20X augmentation. Please click here to view a larger version of this figure.
Figure 2: Protein content of wild-type C. elegans N2. This panel shows the correlation of the number of nematodes to the protein yield of the lysate, prepared as described in protocol section 5 in 12-day-old nematodes(n = 3 lysate of 500 nematodes; n = 3 lysate of 1,000 nematodes; and n = 1 lysate of 1,500 nematodes). The regression line is marked in red (r2 = 0.976, y = 0.64). The total protein concentration was measured using the Bradford method. The data are expressed as the mean ± standard deviation (SD). Please click here to view a larger version of this figure.
Figure 3: Increased formation of fructosyl-lysine after a glucose treatment of C. elegans N2. The concentrations of fructosyl-lysine after a treatment with glucose in 12-day-old nematodes and untreated controls were determined by LC-MS/MS and normalized to a protein concentration determined by the Bradford method. The data are expressed as the mean ± SD. The p-values were determined by Student's t-test, **p < 0.01. The results are from three independent experiments. Please click here to view a larger version of this figure.
Figure 4: Intracellular methylglyoxal concentrations after a methylglyoxal treatment of C. elegans N2. Methylglyoxal concentrations were measured by LC-MS/MS in 12-day-old nematodes treated with methylglyoxal. The samples were collected less than 2 h after the last treatment. The data are expressed as the mean ± SD normalized to the number of nematodes. The p-values were determined by Student's t-test, **p < 0.01. The results are from three independent experiments. Please click here to view a larger version of this figure.
Figure 5: Oxidative stress measured in C. elegans CL2166 after a glucose treatment. Transgenic CL2166 (gst4::GFP)8 containing a glutathione-S-transferase 4-promotor-driven GFP reporter were cultured on 400 µM FUdR plates containing glucose for 2 d. One group was transferred to fresh glucose plates on day 1 as described in section 2 of this protocol. Oxidative stress was measured as an increase in fluorescence [relative light units (RLU)] with a plate reader. The data are expressed as the mean ± SD. The p-values were determined by ANOVA, *p < 0.05. The results are from three independent experiments. Please click here to view a larger version of this figure.
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 population with sodium hypochlorite and sodium hydroxide to destroy the nematodes and harvest the eggs solely. It has to be taken into account that this approach might not be suitable for all experiments. Depending on the ratio of the population size to the volume of the bleaching solution, the harming effect of the bleaching solution influences the development of the embryos15. Especially when studying developmental processes, this could be a crucial step. Concerning the handling of the nematodes, it is crucial to apply as little shear forces as possible throughout the protocol. If not handled with care, a large number of nematodes will perish. For that matter, it is important not to exceed the spinning time and speed. When transferring the nematodes, a cut pipette tip to reduce the number of injured nematodes should be used. For the transfer of the nematodes, the recommended volume of buffer should not be exceeded, as the agar might crack when it becomes too damp, giving the nematodes the opportunity to dig into the plate. While collecting the samples, the M9 buffer must be kept ice-cold to slow down the nematodes' metabolism, especially when working with metabolites or substrates that C. elegans will degrade. It is important to wash the sample thoroughly to minimize any bacterial contamination from prior feeding. When transferring the pellet, remember that a great number of worms could stick to plastic pipette tips. The recommended glass pipette should be used, as even low-binding pipette tips could retain a large amount of the sample. As an alternative, the addition of 0.01% Triton-X100 to the M9 buffer was previously suggested16. To ensure that no deceased C. elegans or bacteria will influence the results, sucrose floatation after the collection of the nematodes can be performed14. This is usually done after liquid culturing to remove the medium. In this experiment, this cleansing was omitted, because sucrose is metabolized into glucose and fructose, thus potentially confounding our results.
Typically, a large-scale culture of C. elegans is performed using liquid media. Liquid culturing, however, is not suitable for all experimental settings, especially when using readouts which require exact numbers of nematodes. A modification of the Baerman apparatus was previously described to sort and purify nematodes derived from liquid culture17. This method greatly reduces bacterial contamination and filters only adult nematodes through a multiple component filter system. This elegant method is limited by the long filtering time (2 – 6 h) at room temperature, which could confound metabolic analyses. As for the filtering technique, nematodes are sorted by their moving activities. Nematodes have to be fit enough to crawl independently through the pores of filters from different materials. Thus, results could be distorted by excluding nematodes that are weakened by the experimental treatments.
Combining liquid and plate cultures in a single experimental design could also serve as a confounder in later analyses. C. elegans develops a thinner and longer phenotype in liquid culture, making it difficult to compare parameters normalized to mass, protein content, or body length and width2. Moreover, the study of reactive substances is challenging in liquid culture, since reactive substances will likely be modified or inactivated by media components before reaching the nematodes. Even though large-scale samples of C. elegans can be obtained with this protocol, plate culture itself is more labor-intensive than liquid culture. However, there are certain ways to facilitate the handling (e.g., with the use of an agar-dispensing machine). Another option to promote large-scale culture efficiently is the use of Petri dishes with a larger diameter for the production of agar plates. This option might be constraint by the succeeding analyses. For microscopic assessment or plate handling in general, the amenities decrease with the diameter of the dish.
Multiple high-throughput approaches suitable for the assessment of different parameters, such as chemical or drug screening, have been developed and investigated18. Microfluidic devices allow researchers to study various parameters, as shown in a model of type-2 diabetes19. Lifespan, lipid metabolism, and oxidative stress responses can be simultaneously assessed on a single-animal level, revealing the advantages of high-content screening, which integrates phenotypic with biochemical information. The assessment of the reliability and reproducibility of the current literature has already shown a great refinement of these techniques, going beyond proof-of-concept studies18. The affordability, especially for smaller laboratories, still remains problematic, as the devices often have to be customized according to experimental needs, which can prove to be costly.
The quantification of AGEs in C. elegans is complicated by the impermeable cuticle of the nematode. A commonly used method is the detection of AGEs via immunofluorescent stainings6. Because of the cuticle, larger samples sizes are needed to decrease the high variance of the results. Imaging has to be taken into account as a time-consuming factor.
The protocol for whole-body lysate preparation described here makes intracellular AGEs and other components of C. elegans accessible for analyses. LC-MS/MS measurements of C. elegans samples have been performed before14. Adequate culturing conditions, however, to achieve a sufficient number of nematodes, have not been described in detail, yet.
The method described in this protocol is useful for readouts requiring a large-scale, homogenously grown population of C. elegans, or for the combination of multiple readouts per experiment. This is particularly convenient for studying complex multifactorial diseases such as diabetes and its complications. Alternative approaches, such as high-throughput and high-content screening using microfluidic systems, are technologically more advanced and are able to provide larger-sized data. Their main application can currently be seen in chemical and drug screening or genetic screening for mutants responding to the experimental treatments. This protocol helps researchers to easily and inexpensively upscale the size of their current or future projects without the need for an adjustment of the current experimental readouts and, thus, to minimize any time loss associated with the establishment of new methods.
The authors have nothing to disclose.
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).
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. |