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Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus
PARINT-01485; No of Pages 4 Parasitology International xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
3Q2
Shinzaburo Takamiya a,b,⁎, Toshihiro Mita a a
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a r t i c l e
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Article history: Received 30 December 2015 Received in revised form 12 March 2016 Accepted 30 March 2016 Available online xxxx
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Department of Molecular and Cellular Parasitology, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Department of Parasitology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
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A method for purifying active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus is described. This method consists of two parts: 1) large-scale cultivation of C. elegans in liquid medium and 2) preparation of active nematodes without contamination using the Baermann apparatus. © 2015 Published by Elsevier Ireland Ltd.
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Keywords: Caenorhabditis elegans Baermann apparatus Liquid culture
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1. Background
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In biochemical studies, it is essential to avoid contamination in the process of preparing the starting materials for the purification of proteins or sub-cellular organelles. When nematodes are starting materials, two types of separation methods based on different principles are used: one utilizes the movement activity of live nematodes, and the other is based on physicochemical properties such as differences in specific gravities between the nematodes and the contaminants. Common contaminants of nematode samples include Escherichia coli, on which Caenorhabditis elegans feeds in the culture medium, and cuticles, which are molted off during nematode development. These materials can be removed by simple or step-wise density gradient centrifugation, utilizing differences in specific gravities between the nematodes and the contaminants, as previously reported for C. elegans [1,2]. Here, we describe a method of large-scale purification of liquid-cultured C. elegans nematodes using a modified Baermann technique, a separation method employing the movement activity of live nematodes.
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2. Objectives
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Baermann's larval technique and its modifications have long been used to detect or recover living nematodes from feces or infective eggs [3–5]. The Ascaris suum infective larvae obtained using this technique
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Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus
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⁎ Corresponding author at: Department of Molecular and Cellular Parasitology, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 1138421, Japan. E-mail address: [email protected] (S. Takamiya).
were successfully cultivated in vitro followed by biochemical analysis of larvae development [6,7], and used to purify the larval complex II [8]. The present method was developed to facilitate comparative mitochondrial proteomics between the parasitic nematode A. suum and the free-living nematode C. elegans, which feeds on E. coli.
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3. Materials and methods
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3.1. Preparation of liquid-cultured C. elegans
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C. elegans (N2 strain) was cultured in S medium using concentrated E. coli OP50 as a food source [9,10]. A concentrated E. coli OP50 pellet was inoculated into 500 mL of S-medium in a sterilized 2-L conical flask. Next, seven large plates containing C. elegans were washed with M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 mL 1 M MgSO4, and H2O to 1 L; sterilized by autoclaving) and the nematodes were transferred into the flask. The flask was incubated at 25 °C with vigorous shaking. Nematode growth was monitored by collecting aliquots of the culture and recording the numbers and stages of development under a light microscope following fixation in formalin solution. When the nematode culture reached log phase (4–6 days) (Note 1), cultivation was stopped and the medium was transferred to a 500-mL cylinder that had been cooled on ice. The cylinder was placed in a cold room for 15 min to allow the nematodes to settle (Note 2), after which most of the supernatant was aspirated from the cylinder. The remaining worm suspension was transferred to a sterile conical centrifuge tube (50 mL) and centrifuged for 2 min at 100 × g to pellet the worms. Finally, the supernatant was removed by aspiration to obtain the dense worm suspension.
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http://dx.doi.org/10.1016/j.parint.2016.03.013 1383-5769/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: S. Takamiya, T. Mita, Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus, Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2016.03.013
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During this time, the nematodes sedimented towards the screw clamp and accumulated near the funnel closure. The Teflon tube was closed using the second screw clamp at the upper boundary of the accumulated worms and the first screw clamp was released to recover the worms (Note 6). The worm suspension was centrifuged at 1770 ×g to obtain a pellet (Note 7), and the pellet was either used immediately for experiments or stored at −80 °C until use.
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3.2.1. Special remarks/comments In our earlier study of C. elegans mitochondria, we cultivated C. elegans in a liquid medium containing E. coli OP50 as food, and purified the samples by decantation and centrifugation according to reported methods [1,2]. However, this preparation method is not appropriate for proteomic analysis of the mitochondria for two reasons. The first reason is that E. coli membrane, derived from contaminated E. coli, was still present in the final mitochondrial preparation, as described below. Such contamination would likely result in additional protein spots on the two-dimensional gel. These should not be included for analysis of in-gel digestion followed by mass spectrometry, but the source of protein spots cannot be distinguished once the sample is contaminated. The other, and more important reason is that decantation or centrifugation cannot distinguish between dead and inactive dying nematodes, which tend to start autolysis during preparation of the mitochondria samples, and thus may result in degraded protein spots on the two-dimensional gel electrophoresis gel. In fact, a significant number of nematodes were found to be immobile among those collected by centrifugation after cultivation. The Baermann techniques are suitable for collecting and concentrating active and alive nematodes selectively, because the separating principle is based on the moving activities of live nematodes. In our laboratories, a prototype of the Baermann apparatus herein described has been used to prepare intact and active A. suum infective larvae, because mechanical hatching of the infective eggs using glass beads unavoidably produces damaged larvae [4].
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A funnel equipped with a stainless steel sieve (mesh No. 16, opening 1 mm, Bunsekifurui Co., Tokyo, Japan) was layered with a sheet of nylon mesh (No. 200), two layers of gauze, and absorbent cotton (Figs. 1, 2) (Note 3). Locke–Ringer's solution (Note 4) was added to the funnel without opening the screw clamp to immerse the layers on the sieve with buffer. The dense worm suspension was spread evenly onto the cotton layer and left to stand for 2–6 h at room temperature (Note 5).
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Fig. 1. Modified Baermann apparatus.
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Fig. 2. Photographs of the modified Baermann apparatus with sheets. (A) Baermann apparatus on an experimental desk. (B) Upper, cotton leaf; middle, two layers of gauze; bottom, nylon sheet (200 mesh). (C) Microscopic photograph of nylon sheet (200 mesh) showing open windows within a 69.4-μm square.
Please cite this article as: S. Takamiya, T. Mita, Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus, Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2016.03.013
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detection of ubiquinone-8 derived from the contaminated E. coli in the preparation before Baermann treatment; however, this was hardly detected by high-performance liquid chromatography of the mitochondrial fraction from the final preparation (Figs. 5, 6). Under the conditions employed (detection limit of ubiquinone-8 in high-performance liquid chromatography was about 0.1 nmol), the molar ratio of ubiquinone-8
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Fig. 5. High performance liquid chromatograms of extracts from the mitochondria of C. elegans before the Baermann technique (top), standard ubiquinones 5, 6, 7, 8, 9, and 10 (middle), and extracts from E. coli, which C. elegans feed on (bottom) (modified from ref. [11]). The striped arrow indicates coexisting rhodoquinone 9 (RQ-9) in C. elegans mitochondria (top). Note a considerable peak (top, left arrow), whose retention time was the same as those for the UQ-8 standard (middle) and for E. coli extracts (bottom, the peak indicated by an arrow). Detailed experimental procedures are described in ref. [11] or [12].
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In this study, we developed a method to collect pure and active C. elegans nematodes, which feed on E. coli, without contamination. The 200-mesh nylon sheet contains 69.4 μm squares, as shown in Fig. 2C, which is shorter than the lengths of nematodes at all developmental stages (L1, 250 μm; L2, 360–380 μm; Dauer, 400 μm; L3, 490– 510 μm; L4, 620–650 μm; Young adult, 900–940 μm; Adult, 1110– 1150 μm) [13] (Note 8). Thus, the worms must crawl into the windows to exit the nylon sheet. Therefore, inactive or dead worms, which are difficult to remove by centrifugation because their specific gravities are similar to those of active nematodes, are expected to remain on the multiple layers of the sieve. In fact, a considerable number of worms and their molted cuticles remained, even after overnight incubation, and were discarded. E. coli, which was supplied as food to the nematodes and was thus a source of contamination can be removed by the current Baermann method. The final mitochondrial preparation contained a slight amount of E. coli membrane, as indicated by the
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Fig. 3. Growth curve of 500 mL liquid culture of Caenorhabditis elegans nematodes (modified from ref. [11]). A lag period was noted in the initial stage of cultivation.
Fig. 4. Photographs of worms recovered from liquid culture of Caenorhabditis elegans. Bars indicate 1 mm. ×40 magnification. (A): Before the Baermann technique. Note that carcass mass can be seen. (B): Final preparation of worms recovered after the Baermann technique.
Please cite this article as: S. Takamiya, T. Mita, Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus, Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2016.03.013
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clamps. The final preparation was free from eggs or cuticles, debris, 163 and carcasses as shown in Fig. 4, and was practically free from E. coli 164 (Figs. 5 and 6). 165 166
Note 8. The idea behind the use of nylon mesh, instead of cotton or linen clothes of conventional Baermann apparatus, originated from the protocol described by Dr. Paul P. Weinstein, who visited our laboratory to transfer the techniques for cultivating A. suum larvae in vitro [7,15]. A similar idea of using metal mesh (opening 50 μm, 300 mesh) was reported in the protocol for the isolation and feeding of Toxocara canis larvae [16].
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Acknowledgments
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Note 7. The yield was 1.7–2.5 mL as a packed volume.
References
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Note 2. The culture was cooled to minimize the movement of the nematodes, which may disturb their settlement.
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Note 3. We used a pipette (1–5 mL, autoclavable BenchMate, Nichiryo Co., Koshigaya-City, Japan) with a plastic chip, the top of which was cut off to form a large outlet so that the dense worm suspension could be pipetted.
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Note 4. A buffered isotonic solution modified for invertebrates containing 8.5 g NaCl, 0.24 g KCl, 0.12 g CaCl2, 0.1 g NaHCO3, and distilled water to a total volume of 1 L. Just before use, Penicillin G (100 international units/mL) and Streptomycin (100 μg/mL) were added to the solution.
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Note 5. Since the apparatus pooled with the Locke–Ringer's solution is heavy, a sturdy and firm stand is recommended to hold it.
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[1] T.J. Fabian, T.E. Johnson, Production of age-synchronous mass cultures of Caenorhabditis elegans, J. Gerontol. Biol. Sci. 49 (1994) B145–B156. [2] L.I. Grad, L.C. Sayles, B.D. Lemire, D. Leister, J.M. Herrmann, Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans, Mitochondria: Practical Protocols, Humana Press, New York, NY 2007, pp. 51–66. [3] Baermann's larval technique, in: Heinz Mehlhorn (Ed.), Encyclopedia of Parasitology, third ed., Vols. 1 A-M, Springer-Verlag, Berlin, Germany 2008, p. 156. [4] J.F. Urban Jr., F.W. Douvres, F.G. Tromba, A rapid method for hatching Ascaris suum eggs in vitro, Proc. Helminthol. Soc. Wash. 48 (1981) 241–243. [5] J.L. Bellaw, M.K. Nielsen, Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures, Vet. Parasitol. 211 (2015) 99–101. [6] F.W. Douvres, J.F. Urban Jr., Factors contributing to the in vitro development of Ascaris suum from second-stage larvae to mature adults, J. Parasitol. 69 (1981) 549–558. [7] S. Takamiya, K. Kita, Wang Hua, P.P. Weinstein, A. Hiraishi, H. Oya, T. Aoki, Developmental changes in the respiratory chain of Ascaris mitochondria, Biochim. Biophys. Acta 1141 (1993) 65–74. [8] F. Saruta, T. Kuramochi, K. Nakamura, S. Takamiya, Y. Yu, T. Aoki, K. Sekimizu, S. Kojima, K. Kita, Stage-specific isoforms of complex II (succinate-ubiquinone oxidoreductase) in mitochondria from the parasitic nematode, Ascaris suum, J. Biol. Chem. 270 (1995) 928–932. [9] J.A. Lewis, J.T. Fleming, Basic culture methods, Methods in Cell Biology, Academic Press, Waltham, MA 1995, pp. 3–29. [10] T. Stierrnagle, Maintenance of C. elegans, in: I.A. Hope (Ed.), C. elegans: A Practical Approach, Oxford University Press, New York 1999, pp. 59–60. [11] S. Takamiya, T. Matsui, H. Taka, K. Murayama, M. Matsuda, T. Aoki, Free-living nematodes Caenorhabditis elegans possess in their mitochondria an additional rhodoquinone, an essential component of the eukaryotic fumarate reductase system, Arch. Biochem. Biophys. 371 (1999) 284–289. [12] T. Matsui, Master's Thesis, Azabu University, School of Environmental Health Sciences: Biochemical Analyses of Mitochondrial Respiratory-chain Components from Parasitic Helminths and the Free-living Nematode Caenorhabditis elegans, 1999 1–24 (in Japanese). [13] A.K. Corsi, Review: a biochemist's guide to Caenorhabditis elegans, Anal. Biochem. 359 (2006) 1–17. [14] S. Takamiya, T. Fujimura, R. Mineki, H. Taka, N. Shindo, T. Mita, S. Takamiya, Preparation of nematodes' mitochondria using the one-step organelle fractionation method: toward mitochondrial proteome analyses, Materials and Methods in Parasitology, Sankei Co., Nagoya 2014, pp. 147–151 (in Japanese). [15] S. Takamiya, Reflection: how parasitic helminths adapt to environmental hypoxia — my wanderings in helminth biochemistry, Juntendo Med. J. 60 (2014) 432–448. [16] N. Akao, A method of preparing Toxocara larval excretory–secretory antigen: how to feed the larvae in vitro, in: S. Takamiya (Ed.), Materials and Methods in Parasitology, Sankei Co., Nagoya 2014, pp. 15–19 (in Japanese).
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Note 1. : A representative growth curve is shown in Fig. 3 (modified from ref. [12]). The time course of cultivation and the ratios of adults, larvae, and eggs in the culture obtained depend on the number of inoculated worms, the amount of bacteria supplied, and the duration of cultivation. A total of 1.2–1.4 × 107 nematodes were grown in 500 mL culture.
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derived from the bacteria was calculated to be less than 0.37% for the mitochondria from the final preparation compared to the 23.3% molar ratio in the sample prior to Baermann treatment. Proteomic analyses of the mitochondria from C. elegans nematodes prepared as described above are currently underway in our laboratories [14].
Note 6. Use of the double screw clamps can avoid unexpected overflow, which would disturb natural sedimentation, and enable the recovery of a second batch of purified worms depending on the experimental purpose. It is convenient to use two angiotribe forceps rather than screw
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This work was supported in part by JSPS KAKENHI Grant Numbers 14570220 and 25460519. We are also indebted to Mr. Kouji Kasanuki and Mr. Toshihiro Matsui for providing technical assistance, and to the anonymous reviewers for the helpful suggestions to revise our manuscript.
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Fig. 6. High performance liquid chromatograms of extracts from the mitochondria of C. elegans after the Baermann technique (top) and standard ubiquinones 5, 6, 7, 8, 9, and 10 (bottom). Note that the ubiquinone-8 derived from contaminated E. coli was almost cleared (top, arrow). Detailed experimental procedures are described in ref. [11] or [12].
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Please cite this article as: S. Takamiya, T. Mita, Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus, Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2016.03.013
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Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
3Q2
Shinzaburo Takamiya a,b,⁎, Toshihiro Mita a a
7
a r t i c l e
8 9 10 11 12 13 18 19 20 21
Article history: Received 30 December 2015 Received in revised form 12 March 2016 Accepted 30 March 2016 Available online xxxx
i n f o
R O
Department of Molecular and Cellular Parasitology, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Department of Parasitology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
a b s t r a c t
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A method for purifying active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus is described. This method consists of two parts: 1) large-scale cultivation of C. elegans in liquid medium and 2) preparation of active nematodes without contamination using the Baermann apparatus. © 2015 Published by Elsevier Ireland Ltd.
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Keywords: Caenorhabditis elegans Baermann apparatus Liquid culture
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In biochemical studies, it is essential to avoid contamination in the process of preparing the starting materials for the purification of proteins or sub-cellular organelles. When nematodes are starting materials, two types of separation methods based on different principles are used: one utilizes the movement activity of live nematodes, and the other is based on physicochemical properties such as differences in specific gravities between the nematodes and the contaminants. Common contaminants of nematode samples include Escherichia coli, on which Caenorhabditis elegans feeds in the culture medium, and cuticles, which are molted off during nematode development. These materials can be removed by simple or step-wise density gradient centrifugation, utilizing differences in specific gravities between the nematodes and the contaminants, as previously reported for C. elegans [1,2]. Here, we describe a method of large-scale purification of liquid-cultured C. elegans nematodes using a modified Baermann technique, a separation method employing the movement activity of live nematodes.
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2. Objectives
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Baermann's larval technique and its modifications have long been used to detect or recover living nematodes from feces or infective eggs [3–5]. The Ascaris suum infective larvae obtained using this technique
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Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus
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⁎ Corresponding author at: Department of Molecular and Cellular Parasitology, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 1138421, Japan. E-mail address: [email protected] (S. Takamiya).
were successfully cultivated in vitro followed by biochemical analysis of larvae development [6,7], and used to purify the larval complex II [8]. The present method was developed to facilitate comparative mitochondrial proteomics between the parasitic nematode A. suum and the free-living nematode C. elegans, which feeds on E. coli.
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3. Materials and methods
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3.1. Preparation of liquid-cultured C. elegans
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C. elegans (N2 strain) was cultured in S medium using concentrated E. coli OP50 as a food source [9,10]. A concentrated E. coli OP50 pellet was inoculated into 500 mL of S-medium in a sterilized 2-L conical flask. Next, seven large plates containing C. elegans were washed with M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 mL 1 M MgSO4, and H2O to 1 L; sterilized by autoclaving) and the nematodes were transferred into the flask. The flask was incubated at 25 °C with vigorous shaking. Nematode growth was monitored by collecting aliquots of the culture and recording the numbers and stages of development under a light microscope following fixation in formalin solution. When the nematode culture reached log phase (4–6 days) (Note 1), cultivation was stopped and the medium was transferred to a 500-mL cylinder that had been cooled on ice. The cylinder was placed in a cold room for 15 min to allow the nematodes to settle (Note 2), after which most of the supernatant was aspirated from the cylinder. The remaining worm suspension was transferred to a sterile conical centrifuge tube (50 mL) and centrifuged for 2 min at 100 × g to pellet the worms. Finally, the supernatant was removed by aspiration to obtain the dense worm suspension.
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http://dx.doi.org/10.1016/j.parint.2016.03.013 1383-5769/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: S. Takamiya, T. Mita, Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus, Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2016.03.013
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During this time, the nematodes sedimented towards the screw clamp and accumulated near the funnel closure. The Teflon tube was closed using the second screw clamp at the upper boundary of the accumulated worms and the first screw clamp was released to recover the worms (Note 6). The worm suspension was centrifuged at 1770 ×g to obtain a pellet (Note 7), and the pellet was either used immediately for experiments or stored at −80 °C until use.
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3.2.1. Special remarks/comments In our earlier study of C. elegans mitochondria, we cultivated C. elegans in a liquid medium containing E. coli OP50 as food, and purified the samples by decantation and centrifugation according to reported methods [1,2]. However, this preparation method is not appropriate for proteomic analysis of the mitochondria for two reasons. The first reason is that E. coli membrane, derived from contaminated E. coli, was still present in the final mitochondrial preparation, as described below. Such contamination would likely result in additional protein spots on the two-dimensional gel. These should not be included for analysis of in-gel digestion followed by mass spectrometry, but the source of protein spots cannot be distinguished once the sample is contaminated. The other, and more important reason is that decantation or centrifugation cannot distinguish between dead and inactive dying nematodes, which tend to start autolysis during preparation of the mitochondria samples, and thus may result in degraded protein spots on the two-dimensional gel electrophoresis gel. In fact, a significant number of nematodes were found to be immobile among those collected by centrifugation after cultivation. The Baermann techniques are suitable for collecting and concentrating active and alive nematodes selectively, because the separating principle is based on the moving activities of live nematodes. In our laboratories, a prototype of the Baermann apparatus herein described has been used to prepare intact and active A. suum infective larvae, because mechanical hatching of the infective eggs using glass beads unavoidably produces damaged larvae [4].
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A funnel equipped with a stainless steel sieve (mesh No. 16, opening 1 mm, Bunsekifurui Co., Tokyo, Japan) was layered with a sheet of nylon mesh (No. 200), two layers of gauze, and absorbent cotton (Figs. 1, 2) (Note 3). Locke–Ringer's solution (Note 4) was added to the funnel without opening the screw clamp to immerse the layers on the sieve with buffer. The dense worm suspension was spread evenly onto the cotton layer and left to stand for 2–6 h at room temperature (Note 5).
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Fig. 1. Modified Baermann apparatus.
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Fig. 2. Photographs of the modified Baermann apparatus with sheets. (A) Baermann apparatus on an experimental desk. (B) Upper, cotton leaf; middle, two layers of gauze; bottom, nylon sheet (200 mesh). (C) Microscopic photograph of nylon sheet (200 mesh) showing open windows within a 69.4-μm square.
Please cite this article as: S. Takamiya, T. Mita, Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus, Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2016.03.013
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detection of ubiquinone-8 derived from the contaminated E. coli in the preparation before Baermann treatment; however, this was hardly detected by high-performance liquid chromatography of the mitochondrial fraction from the final preparation (Figs. 5, 6). Under the conditions employed (detection limit of ubiquinone-8 in high-performance liquid chromatography was about 0.1 nmol), the molar ratio of ubiquinone-8
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Fig. 5. High performance liquid chromatograms of extracts from the mitochondria of C. elegans before the Baermann technique (top), standard ubiquinones 5, 6, 7, 8, 9, and 10 (middle), and extracts from E. coli, which C. elegans feed on (bottom) (modified from ref. [11]). The striped arrow indicates coexisting rhodoquinone 9 (RQ-9) in C. elegans mitochondria (top). Note a considerable peak (top, left arrow), whose retention time was the same as those for the UQ-8 standard (middle) and for E. coli extracts (bottom, the peak indicated by an arrow). Detailed experimental procedures are described in ref. [11] or [12].
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In this study, we developed a method to collect pure and active C. elegans nematodes, which feed on E. coli, without contamination. The 200-mesh nylon sheet contains 69.4 μm squares, as shown in Fig. 2C, which is shorter than the lengths of nematodes at all developmental stages (L1, 250 μm; L2, 360–380 μm; Dauer, 400 μm; L3, 490– 510 μm; L4, 620–650 μm; Young adult, 900–940 μm; Adult, 1110– 1150 μm) [13] (Note 8). Thus, the worms must crawl into the windows to exit the nylon sheet. Therefore, inactive or dead worms, which are difficult to remove by centrifugation because their specific gravities are similar to those of active nematodes, are expected to remain on the multiple layers of the sieve. In fact, a considerable number of worms and their molted cuticles remained, even after overnight incubation, and were discarded. E. coli, which was supplied as food to the nematodes and was thus a source of contamination can be removed by the current Baermann method. The final mitochondrial preparation contained a slight amount of E. coli membrane, as indicated by the
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Fig. 3. Growth curve of 500 mL liquid culture of Caenorhabditis elegans nematodes (modified from ref. [11]). A lag period was noted in the initial stage of cultivation.
Fig. 4. Photographs of worms recovered from liquid culture of Caenorhabditis elegans. Bars indicate 1 mm. ×40 magnification. (A): Before the Baermann technique. Note that carcass mass can be seen. (B): Final preparation of worms recovered after the Baermann technique.
Please cite this article as: S. Takamiya, T. Mita, Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus, Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2016.03.013
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clamps. The final preparation was free from eggs or cuticles, debris, 163 and carcasses as shown in Fig. 4, and was practically free from E. coli 164 (Figs. 5 and 6). 165 166
Note 8. The idea behind the use of nylon mesh, instead of cotton or linen clothes of conventional Baermann apparatus, originated from the protocol described by Dr. Paul P. Weinstein, who visited our laboratory to transfer the techniques for cultivating A. suum larvae in vitro [7,15]. A similar idea of using metal mesh (opening 50 μm, 300 mesh) was reported in the protocol for the isolation and feeding of Toxocara canis larvae [16].
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Acknowledgments
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Note 7. The yield was 1.7–2.5 mL as a packed volume.
References
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Note 2. The culture was cooled to minimize the movement of the nematodes, which may disturb their settlement.
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Note 3. We used a pipette (1–5 mL, autoclavable BenchMate, Nichiryo Co., Koshigaya-City, Japan) with a plastic chip, the top of which was cut off to form a large outlet so that the dense worm suspension could be pipetted.
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Note 4. A buffered isotonic solution modified for invertebrates containing 8.5 g NaCl, 0.24 g KCl, 0.12 g CaCl2, 0.1 g NaHCO3, and distilled water to a total volume of 1 L. Just before use, Penicillin G (100 international units/mL) and Streptomycin (100 μg/mL) were added to the solution.
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Note 5. Since the apparatus pooled with the Locke–Ringer's solution is heavy, a sturdy and firm stand is recommended to hold it.
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[1] T.J. Fabian, T.E. Johnson, Production of age-synchronous mass cultures of Caenorhabditis elegans, J. Gerontol. Biol. Sci. 49 (1994) B145–B156. [2] L.I. Grad, L.C. Sayles, B.D. Lemire, D. Leister, J.M. Herrmann, Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans, Mitochondria: Practical Protocols, Humana Press, New York, NY 2007, pp. 51–66. [3] Baermann's larval technique, in: Heinz Mehlhorn (Ed.), Encyclopedia of Parasitology, third ed., Vols. 1 A-M, Springer-Verlag, Berlin, Germany 2008, p. 156. [4] J.F. Urban Jr., F.W. Douvres, F.G. Tromba, A rapid method for hatching Ascaris suum eggs in vitro, Proc. Helminthol. Soc. Wash. 48 (1981) 241–243. [5] J.L. Bellaw, M.K. Nielsen, Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures, Vet. Parasitol. 211 (2015) 99–101. [6] F.W. Douvres, J.F. Urban Jr., Factors contributing to the in vitro development of Ascaris suum from second-stage larvae to mature adults, J. Parasitol. 69 (1981) 549–558. [7] S. Takamiya, K. Kita, Wang Hua, P.P. Weinstein, A. Hiraishi, H. Oya, T. Aoki, Developmental changes in the respiratory chain of Ascaris mitochondria, Biochim. Biophys. Acta 1141 (1993) 65–74. [8] F. Saruta, T. Kuramochi, K. Nakamura, S. Takamiya, Y. Yu, T. Aoki, K. Sekimizu, S. Kojima, K. Kita, Stage-specific isoforms of complex II (succinate-ubiquinone oxidoreductase) in mitochondria from the parasitic nematode, Ascaris suum, J. Biol. Chem. 270 (1995) 928–932. [9] J.A. Lewis, J.T. Fleming, Basic culture methods, Methods in Cell Biology, Academic Press, Waltham, MA 1995, pp. 3–29. [10] T. Stierrnagle, Maintenance of C. elegans, in: I.A. Hope (Ed.), C. elegans: A Practical Approach, Oxford University Press, New York 1999, pp. 59–60. [11] S. Takamiya, T. Matsui, H. Taka, K. Murayama, M. Matsuda, T. Aoki, Free-living nematodes Caenorhabditis elegans possess in their mitochondria an additional rhodoquinone, an essential component of the eukaryotic fumarate reductase system, Arch. Biochem. Biophys. 371 (1999) 284–289. [12] T. Matsui, Master's Thesis, Azabu University, School of Environmental Health Sciences: Biochemical Analyses of Mitochondrial Respiratory-chain Components from Parasitic Helminths and the Free-living Nematode Caenorhabditis elegans, 1999 1–24 (in Japanese). [13] A.K. Corsi, Review: a biochemist's guide to Caenorhabditis elegans, Anal. Biochem. 359 (2006) 1–17. [14] S. Takamiya, T. Fujimura, R. Mineki, H. Taka, N. Shindo, T. Mita, S. Takamiya, Preparation of nematodes' mitochondria using the one-step organelle fractionation method: toward mitochondrial proteome analyses, Materials and Methods in Parasitology, Sankei Co., Nagoya 2014, pp. 147–151 (in Japanese). [15] S. Takamiya, Reflection: how parasitic helminths adapt to environmental hypoxia — my wanderings in helminth biochemistry, Juntendo Med. J. 60 (2014) 432–448. [16] N. Akao, A method of preparing Toxocara larval excretory–secretory antigen: how to feed the larvae in vitro, in: S. Takamiya (Ed.), Materials and Methods in Parasitology, Sankei Co., Nagoya 2014, pp. 15–19 (in Japanese).
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Note 1. : A representative growth curve is shown in Fig. 3 (modified from ref. [12]). The time course of cultivation and the ratios of adults, larvae, and eggs in the culture obtained depend on the number of inoculated worms, the amount of bacteria supplied, and the duration of cultivation. A total of 1.2–1.4 × 107 nematodes were grown in 500 mL culture.
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derived from the bacteria was calculated to be less than 0.37% for the mitochondria from the final preparation compared to the 23.3% molar ratio in the sample prior to Baermann treatment. Proteomic analyses of the mitochondria from C. elegans nematodes prepared as described above are currently underway in our laboratories [14].
Note 6. Use of the double screw clamps can avoid unexpected overflow, which would disturb natural sedimentation, and enable the recovery of a second batch of purified worms depending on the experimental purpose. It is convenient to use two angiotribe forceps rather than screw
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This work was supported in part by JSPS KAKENHI Grant Numbers 14570220 and 25460519. We are also indebted to Mr. Kouji Kasanuki and Mr. Toshihiro Matsui for providing technical assistance, and to the anonymous reviewers for the helpful suggestions to revise our manuscript.
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Fig. 6. High performance liquid chromatograms of extracts from the mitochondria of C. elegans after the Baermann technique (top) and standard ubiquinones 5, 6, 7, 8, 9, and 10 (bottom). Note that the ubiquinone-8 derived from contaminated E. coli was almost cleared (top, arrow). Detailed experimental procedures are described in ref. [11] or [12].
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Please cite this article as: S. Takamiya, T. Mita, Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus, Parasitology International (2015), http://dx.doi.org/10.1016/j.parint.2016.03.013
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