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

Summary

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.

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.

Introduction

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.

Protocol

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

1. In general, work under a sterile hood or next to an open flame to avoid contaminations.
2. To culture worms until they reach the young adult stage, follow this previously published protocol⁷ with the following adaptations.
   1. Use living Escherichia coli OP50 bacteria to feed the nematodes and prepare 400 µM fluorodeoxyuridine agar (not ampicillin/FUdR) for experiments.
   2. For the preparation of a synchronous population, use unconcentrated OP50, and for succeeding experiments with the adults, use 3.3x concentrated OP50 by removing 70% of the supernatant.
   3. Daily feeding intervals are recommended. When assessing oxidative stress, different intervals and volumes for feeding might interfere with the results.
   4. For inoculation, take a plate with several nematodes and cut it into pieces of approximately 0.5 x 1 cm² with a scalpel. Transfer two to three pieces onto nematode growth media (NGM) plates of 60 mm in diameter, containing unconcentrated OP50.
   5. Incubate the plates at the temperature suitable for the strain (wild-type N2 should be cultured at 20 °C) until eggs are visible on the plate.
   6. Wash off most of the eggs and adult animals with 2 mL of M9 buffer and collect them in a 15 mL tube. Centrifuge the suspension at 800 x g for 2 min. Meanwhile, start preparing the bleaching solution.
   7. Take out the centrifuged tube and remove the M9 buffer with a 10 mL pipet. Add 10 mL of the bleaching solution and mix on the vortex until the adult nematodes are lysed. This takes usually approx. 5–7 min, but should not take longer than 15 min or the eggs will not develop fully later on in the experiment.
   8. Centrifuge the suspension at 800 x g for 2 min. Wash 3 times with 10 mL M9 buffer to clear the eggs from bleaching solution.
   9. Now add 150 µL of the egg suspension on OP50 plates. A density of 200–300 eggs should be reached on each plate. Wait until nematodes reached the adult stage.
      NOTE: The M9 buffer (pH 7.0) used for the depicted experiments consists of the following substances: 22 mM KH₂PO₄, 42 mM Na₂HPO₄, and 86 mM NaCl. After autoclaving the mixture, add 1 mM MgSO4. The bleaching solution contains 0.5 M NaOH, 2.3 mM NaOCl solved in distilled water.
   10. Prepare sterile M9 buffer, sterile pipette tips with a cut tip to wash off the worms, and 15 mL tubes to collect them in.
   11. Apply approximately 1 mL of M9 buffer on the plates, gently distribute the buffer by swinging and gently pipet the nematodes off the plate.
   12. Now, collect the nematodes in the tube. Apply 150 µL of suspension directly on the plates (seeded with OP50) with the cut pipette tips, and evaluate microscopically the density of the nematodes (recommended: 50–100 nematodes per 60 mm plate).
   13. If they are too dense, dilute the suspension with M9 buffer and evaluate it under the microscope until the desired density is reached. If the yield is too little, let the worms settle down or centrifuge the suspension 10 s at 800 x g to remove fluid.
       NOTE: An example of a sufficient density is given in Figure 1A (macroscopic) and 1B (microscopic) at 20X augmentation. Proceed empirically as described.
   14. Keep in mind that the worms settle down quickly. To ensure an equal distribution between the plates, invert the tube after every stack of plates (4–5 plates).
       NOTE: The cut pipette tip, as well as the gentle swinging and washing of the plates, reduces shear stress.

2. Transfer of Large Amounts of Caenorhabditis elegans

1. Wash off the nematodes with approximately 500 µL of M9 buffer, depending on the age and dryness of the plates. Using the same buffer, repeat detaching the nematodes until most of the animals are collected in suspension.
2. Slowly take up the nematodes with a cut pipette tip and transfer them on a plate prepared with OP50. Let the plate dry.
   NOTE: Be careful not to apply much more than the recommended volume. Especially in long-term experiments, the plates might not tolerate it and the agar can break, allowing the nematodes to crawl into the cracks.

3. Harvest of a Large Caenorhabditis elegans Sample

1. Depending on the selected method, start experiments with a sufficient amount of C. elegans. For LC-MS/MS analyses, prepare 20 plates (60 mm x 15 mm) per group with approximately 100 nematodes per plate to ensure a yield of at least 200 µg of protein per sample.
   NOTE: This part of the protocol does not require working under sterile conditions anymore, as the nematodes will be collected and frozen. 
2. On the day of the sample collection, prepare liquid nitrogen to snap-freeze the collected samples. Keep non-sterile M9 buffer on ice to slow down the nematode’s metabolism.
3. Wash off the C. elegans from the plates by applying 1 mL of M9 buffer and gently swirling the plates. This avoids collecting deceased nematodes and most eggs, if there are any present, because they stick to the bacteria and agar.
4. Take up the volume and transfer it to a 15 mL tube.
5. After the collection of the complete sample, wait for the nematodes to settle or centrifuge the tube briefly at 800 x g and remove most of the M9 buffer. Wash the sample 3x with approximately 10 mL of M9 (10x the sample volume) to remove any bacteria.
   NOTE: To ensure that all deceased nematodes are removed, sucrose floatation is another option. However, this was not suitable for the displayed experiments¹⁵. Sucrose is hydrolyzed to glucose and fructose. In the depicted experiments, the role of glucose was assessed to promote biochemical changes found in chronic diabetes. Thus, sucrose floatation could potentially confound the results.
6. Prepare one 1.5 mL tube with 1 mL of M9 buffer and one empty 1.5 mL tube for every sample. Transfer the pellet with a glass pipet from the 15 mL tube to the empty 1.5 mL tube. This step is crucial to ensure a quantitative transfer.
   NOTE: Unlike during previous steps, here the nematodes are more concentrated. Because of a possible adherence to plastic pipet tips, loss of a large part of the sample is expected. Therefore, use glass pipettes instead.
7. After the transfer, use the glass pipette to take up 1 mL of M9 buffer from the second 1.5 mL tube to rinse the nematodes from the pipette wall. Also, wash the remaining nematodes from the 15 mL tube and transfer them to the sample tube.
8. Centrifuge the sample tube at 19,000 x g for 1 min and then remove as much supernatant as possible.
9. Seal the tube with Parafilm and immediately snap-freeze it in liquid nitrogen. Store the tube at -80 °C until the lysate preparation.

4. Protein Isolation for Mass Spectrometry

NOTE: The protein isolation described here can also be used for other assays (e.g., western blot).

1. To the frozen sample, add the same volumes of 0.5 mm zirconium oxide beads and at least 2x the volume of lysate buffer containing a proteinase inhibitor cocktail.
   NOTE: The depicted nematodes-to-protein correlation analyses were performed using 100 µL of buffer. LC-MS/MS samples were lysed with 200 µL of buffer.
2. Start the homogenizer for 1 min on speed 9. Ensure that the samples are completely lysed. Repeat this step if they are not fully lysed.
3. Centrifuge the samples for 30 min at 4 °C at 19,000 x g. Transfer the supernatant to a new tube on ice and store it at -80 °C until further measurements are going to be taken.
   NOTE: The non-denaturing buffer used for the depicted experiments consists of the following substances: 20 mM Tris HCl (pH 8), 137 mM NaCl, 1% Triton-X, and 2 mM EDTA.
   NOTE: Having followed the steps of this protocol and having applied the suggested scale, the yield of a protein pellet from approximately 1,000 nematodes should be approximately 500 µg. For more comparisons, consult Figure 2 and the Representative Results.

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.

1. Measure intracellular methylglyoxal by derivatization with 1,2-diaminobenzene (DB) as previously described⁹.
   1. Homogenize the samples with 20 µL of ice-cold 20% (wt/vol) trichloroacetic acid in 0.9% (wt/vol) sodium chloride and 80 µL of water in a 1.5 mL tube.
   2. Spike the samples of 5 µL with the internal standard (¹³C₃-MG; 400 nM), mix them by vortexing, and then centrifuge them at 21,000 x g for 5 min at 4 °C.
   3. Transfer 35 µL of the supernatant to the HPLC sample vial containing a 200 µL glass insert.
   4. Add 5 µL of 3% sodium azide (wt/vol) to each sample followed by 10 µL of 0.5 mM DB in 200 mM HCl containing 0.5 mM diethylenetriaminepentaacetic acid (DETAPAC) in water.
   5. Incubate the samples for 4 h at room temperature, protected from light.
   6. Analyze the samples by LC-MS/MS, as previously described⁹.
      NOTE: For the measurement of AGEs, isolate the proteins as described in section 4 of the protocol.
2. Measure the protein concentration of the (thawed) lysates using a Bradford assay¹⁰.
   1. Prepare aliquots of 30 µL for a standard curve spiked with bovine serum albumin (BSA) solved in phosphate-buffered saline (PBS) of the following concentration: 0; 0.1; 0.2; 0.3; 0.4; 0.6; 0.8; and 1.0 mg/dL.
      NOTE: If the sample was prepared at the suggested size (see step 4.1 for the preparation of lysate with 200 µL of buffer), the estimated concentration should be in the range of approximately 2–7 mg/mL. In this case, dilute the samples 1:10 with PBS prior to the measurement.
      NOTE: For the first experiments using this method, it is recommended to measure the samples in different dilutions (1, 1:10, and 1:100). As there is no exact determination of the number of nematodes, the yield can differ between individual researchers.
   2. Use a transparent 96-well plate (polystyrene) and pipette 10 µL of each standard concentration and of the samples in the chosen dilutions in duplicates in the wells.
   3. Dilute the protein assay dye reagent concentrate 1:5 with distilled water (aq. dest.) and add 200 µL of the dilution to every sample on the plate. Incubate the plate for 10 min at room temperature.
   4. Measure the absorbance within 30 min at 595 nm with a plate reader. The samples can be interpolated from the calculated standard curve. More details of the method have been described extensively in the literature¹⁰.
3. Measure the intracellular AGEs as previously described^11,12.
   1. Wash the total protein extracts (approximately 200 µg) 3x with water by ultracentrifugation in 10 kDa centrifugal filter units.
   2. Add 10 µL of 100 mM HCl, 10 µL of pepsin (2 mg/mL in 20 mM HCl), and 10 µL of thymol (2 mg/mL in 20 mM HCl) to the recovered protein (approximately 100 µL) and incubate the samples at 37 °C for 24 h.
   3. Neutralize and buffer the samples at pH 7.4 by adding 12.5 µL of 0.5 M potassium phosphate buffer (pH 7.4) and 5 µL of 260 mM KOH.
   4. Add 10 µL of Pronase E [2 mg/mL in 10 mM potassium phosphate buffer (pH 7.4)] and 10 µL of a penicillin-streptomycin solution, and incubate the samples at 37 °C for 24 h.
   5. Add 10 µL of aminopeptidase [2 mg/mL in 10 mM potassium phosphate buffer (pH 7.4)] and 10 µL of prolidase [2 mg/mL in 10 mM potassium phosphate buffer (pH 7.4)], and incubate the samples at 37 °C for 48 h.
   6. Analyze the resulting hydrolysate by LC-MS/MS, as previously described¹².

Results

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...

Discussion

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...

Materials

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 mm 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.