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

Abstract

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.

Protective Effects of Transient Glucose Exposure in Adult C. elegans

“…Multiple steps of physical, chemical and enzymatic treatment were necessary to isolate and unmask the AGEs argpyrimidine (AP), fructosyllysine (FL) and methylglyoxal-hydroimidazolone-1 (MG-H1) [ 4 , 5 ]. These were then measured using liquid chromatography–tandem mass spectrometry (LC-MS/MS) [ 32 , 33 ]. Briefly, protein extracts were purified by ultracentrifugation and digested by pepsin and thymol in the presence of HCl.…”

“…Briefly, protein extracts were purified by ultracentrifugation and digested by pepsin and thymol in the presence of HCl. After buffering and further degradation by pronase and aminopeptidase the unmasked AGEs were then measured using liquid chromatography–tandem mass spectrometry (LC-MS/MS) [ 32 , 33 ].…”

Protein Extract Preparation and Co-immunoprecipitation from <em>Caenorhabditis elegans</em>

“…NOTE: The amount of antibody used for immunoprecipitation is specific to the antibody and protein and should be empirically determined to ensure effective immunoprecipitation of the target protein. 8. Add the remaining 75 µL of the prewashed beads suspension (step 3.5) to the antibody/lysate mixture and incubate for 1 h at 4 °C with gentle agitation.…”

“…Efficient lysis of the sample is necessary, but care must be taken to minimize the disruption of protein-protein interactions. Methods such as douncing 5 , sonication 6 , Balch homogenization 7 , and zirconia beads-homogenization 8,9 have been used to successfully prepare C. elegans total protein extracts. These methods, with the exception of zirconia bead homogenization, have limitations in terms of the number of samples that can be processed simultaneously.…”

“…This extract protocol allows for rapid protein extract preparation from C. elegans samples and is comparable to zirconium bead-based homogenization 8 . Bead homogenization in general can be scaled up to multiple simultaneous sample preparations using a variety of bead mill homogenizers or similar equipment.…”