Needle wounding methods for C. elegans were first developed by Nathalie Pujol and Jonathan Ewbank; we thank Nathalie Pujol for comments on the manuscript. Work in our laboratory on C. elegans epidermal wound repair is supported by a grant from the NIH to A.D.C. (R01 GM054657).

<ol>
	<li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li><li>Sonnemann, K. J., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair:+toward+understanding+and+integration+of+single-cell+and+multicellular+wound+responses.">Wound repair: toward understanding and integration of single-cell and multicellular wound responses.</a> Annu Rev Cell Dev Biol. 27, 237-263 (2011).</li><li>Xu, S., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+G&#945;q-Ca2++signaling+pathway+promotes+actin-mediated+epidermal+wound+closure+in+C.+elegans.">A G&#945;q-Ca2+ signaling pathway promotes actin-mediated epidermal wound closure in C. elegans.</a> Curr Biol. 21, 1960-1967 (2011).</li><li>Pujol, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+innate+immune+responses+to+infection+and+wounding+in+the+C.+elegans+epidermis.">Distinct innate immune responses to infection and wounding in the C. elegans epidermis.</a> Curr Biol. 18, 481-489 (2008).</li><li>Chisholm, A. 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Methods in cell biology.</a> 48, 225-250 .</li><li>Pujol, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-fungal+innate+immunity+in+C.+elegans+is+enhanced+by+evolutionary+diversification+of+antimicrobial+peptides.">Anti-fungal innate immunity in C. elegans is enhanced by evolutionary diversification of antimicrobial peptides.</a> PLoS Pathog. 4, (2008).</li><li>Tong, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+regulation+of+Caenorhabditis+elegans+epidermal+damage+responses+by+death-associated+protein+kinase.">Negative regulation of Caenorhabditis elegans epidermal damage responses by death-associated protein kinase.</a> Proc Natl Acad Sci U S A. 106, 1457-1461 (2009).</li><li>Zugasti, O., Ewbank, J. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+Conidia+of+Drechmeria+coniospora+to+Caenorhabditis+elegans+Wild+Type+and+Mutants.">Adhesion of Conidia of Drechmeria coniospora to Caenorhabditis elegans Wild Type and Mutants.</a> J Nematol. 26, 430-435 (1994).</li><li>Hodgkin, J., Kuwabara, P. E., Corneliussen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+bacterial+pathogen,+Microbacterium+nematophilum,+induces+morphological+change+in+the+nematode+C.+elegans.">A novel bacterial pathogen, Microbacterium nematophilum, induces morphological change in the nematode C. elegans.</a> Curr Biol. 10, 1615-1618 (2000).</li><li>Hochbaum, D., Ferguson, A. A., Fisher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transgenic+C.+elegans+by+biolistic+transformation.">Generation of transgenic C. elegans by biolistic transformation.</a> J Vis Exp. (42), (2010).</li><li>Hashmi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+nematodes+using+arrays+of+micromechanical+piercing+structures.">Genetic transformation of nematodes using arrays of micromechanical piercing structures.</a> BioTechniques. 19, 766-770 (1995).</li></ol>

The authors are not aware of any competing interests.

الأساليب المقدمة هنا للإبرة وجرح الليزر توفر نهج تكميلية لتقييم قدرة ظهارة البشرة لإصلاح الضرر. جرح الليزر محلية نسبيا و(اعتمادا على تكوين ليزر) يمكن أن تقتصر على البشرة، في حين جرح إبرة يعطل البشرة، بشرة، والمرجح الأغشية الطابق السفلي الداخلية. إبرة جرح قد تشبه بشك?...

آليات الشفاء الجلد الجروح ذات أهمية بيولوجية أساسية وذات الصلة بصحة الإنسان. ويضم التئام الجروح في الفقاريات والثدييات سلسلة معقدة من استجابات منسقة من أنسجة متعددة ويشير 1. كثير من الكائنات نموذج الجيني بسيطة هي أيضا قادرة على شفاء الجروح الجلدية 2. ولذلك من مصلحة لتحليل نماذج الانقياد وراثيا من إصلاح الجرح الجلد. نحن وغيرنا قد بدأت في استخدام انواع معينة ايليجانس نموذجا جديدا للشفاء الجرح الجلد 3،4. والهدف من هذا البروتوكول هو تمكين مجموعة أوسع من الباحثين لاستخدام C. ايليجانس كأداة للتحقيق في الآليات الجزيئية والخلوية من البشرة التئام الجروح. وC. ويضم ايليجانس الجلد البشرة (المعروف أيضا باسم اللحمة) وبشرة خارج الخلية 5. يتم تشكيل البشرة الكبار من عدد صغير من syncytia متعدد النوى، والتي من أكبر هو مخلى كnown كما hyp7. البشرة هي ظهارة البسيطة التي تفرز بشرة على سطحه قمي. الجلد يمكن أن يدافع بنشاط ضد مسببات الأمراض اختراق الجلد وإصلاح الجروح الصغيرة (4). إصلاح الجرح من C. ايليجانس الجلد هو قوي، وتقريبا جميع الحيوانات البرية نوع البقاء على قيد الحياة يمكن البقاء على قيد الحياة الجروح الصغيرة الناجمة عن الإبر، أو تلف الجلد المحلية التي تسببها أشعة الليزر. C. ايليجانس جرح الجلد مشغلات مجموعة من الردود، بما في ذلك البشرة استجابة فطرية المناعة، وإغلاق الجروح، وتشكيل ندبة 4. البشرة البالغين في مرحلة ما بعد الإنقسامية، وينطوي على التئام الجروح الاستجابات الخلوية المحلية في مقابل انتشار البشرة أو الهجرة الخلية. لقد أثبتنا أن جرح الجلد يتسبب في زيادة كبيرة ومطردة في البشرة الكالسيوم 2+، الأمر الذي يتطلب قناة TRPM غشاء تحويل الغاز إلى سوائل-2 والداخلية الكالسيوم 2+ مخازن 3. مطلوب البشرة الكالسيوم 2+ إشارة لتشكيل وإغلاق حلقات F-أكتين في التعليم الجامعيالموقع اوند. كما يدفع جرح الاستجابات المناعية الفطرية التي تنشط النسخ من المكاتب الإقليمية للمرأة مثل البرمجة اللغوية العصبية-29. النسخ الناجم عن جرح المكاتب الإقليمية للمرأة يعتمد على TIR-1 / PMK-1 P38 MAP كيناز سلسلة يتصرف بشكل مستقل في البشرة 4. سوف العيوب سواء في إشارة مسار الكالسيوم 2+ أو في الاستجابة المناعية الفطرية يؤدي إلى انخفاض البقاء على قيد الحياة بعد إصابة. هيكل بسيط نسبيا من C. ايليجانس البشرة، قابلية الإستطراق الجيني والمزايا لفي الجسم الحي التصوير تجعل من نظام ممتاز لدراسة جوانب متعددة من إصلاح الجرح. هنا نقدم بروتوكولات الطرق الشائعة اثنين من جرح: جرح إبرة وجرح الليزر. جرح إبرة لا يتطلب أي معدات متخصصة (غير مجتذب إبرة)، ومع خبرة لا يمكن أن يؤديها على مئات من الديدان يوميا. يتم تنفيذ جرح إبرة على الحيوانات التي تنمو على لوحات أجار. في المقابل، يتم إجراء جرح الليزر على أنيسالحيوانات thetized التي شنت على أجار منصات تحت ساترة، ومناسب للتصوير المباشر لالاستجابات الخلوية للتلف.

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			<td height="22" style="height:22px;width:227px;">Name</td>
			<td style="width:227px;">Company</td>
			<td style="width:227px;">Catalog Number</td>
			<td style="width:227px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Agarose</td>
			<td style="width:227px;">Denville Scientific</td>
			<td style="width:227px;">CA3510-8</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Plastic Petri plate</td>
			<td style="width:227px;">Tritech Research</td>
			<td style="width:227px;">T3308</td>
			<td style="width:227px;">60 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Levamisole</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:227px;">L9756</td>
			<td style="width:227px;">12 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;"><a name="RANGE!A5">Borosilicate Glass Capillary&#160;</a></td>
			<td style="width:227px;">World Precision Instruments</td>
			<td style="width:227px;">1B100F-4</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Needle puller</td>
			<td style="width:227px;">Sutter Instrument&#160;</td>
			<td style="width:227px;">P-2000</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Worm pick (Platinum wire)</td>
			<td style="width:227px;">Tritech Research</td>
			<td style="width:227px;">PT-9010</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="86">
			<td height="86" style="height:87px;width:227px;">M9 solution</td>
			<td style="width:227px;">Home-made</td>
			<td colspan="2" style="width:453px;">3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Sterilize by autoclaving</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Trizol</td>
			<td style="width:227px;">Invitrogen</td>
			<td align="right" style="width:227px;">15596026</td>
			<td style="width:227px;">Use 500 ml for 50 worms</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">iQ SYBR Green</td>
			<td style="width:227px;">Bio-Rad</td>
			<td align="right" style="width:227px;">1708882</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">SuperScript III</td>
			<td style="width:227px;">Invitrogen</td>
			<td style="width:227px;">18080-051</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">PCR machine</td>
			<td style="width:227px;">Bio-Rad</td>
			<td style="width:227px;">C1000/CFX96</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">96-well PCR tubes</td>
			<td style="width:227px;">Bio-Rad</td>
			<td style="width:227px;">MLL9601</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">Image analysis software</td>
			<td style="width:227px;">Molecular Devices</td>
			<td style="width:227px;">Metamorph</td>
			<td style="width:227px;"></td>
		</tr>
	</tbody>
</table>

methods for skin wounding and assays for wound responses in c. elegans

وC. ايليجانس البشرة وبشرة تشكل طبقة الجلد بعد متطورة البسيطة التي يمكن إصلاح الأضرار المحلية الناجمة عن اصابة. لقد أضاءت الدراسات الردود الجرح وإصلاح في هذا النموذج فهمنا للهيكل الخلية وردود الجينومية إلى تلف الأنسجة. طريقتين الأكثر استخداما لالجرح C. ايليجانس الجلد البالغين من الغرزات مع الإبر حقن مكروي، وأشعة الليزر المحلي. إبرة اصابة محليا يعطل بشرة، البشرة، وما يرتبط بها من المصفوفة خارج الخلية، وأيضا قد تضر الأنسجة الداخلية. النتائج أشعة الليزر في الضرر أكثر محلية. جرح يتسبب في سلسلة من ردود يعاير بسهولة بما في ذلك البشرة مرتفعة الكالسيوم 2+ (ثواني دقيقة)، وتشكيل وإغلاق حلقة التي تحتوي على الأكتين في موقع الجرح (1-2 ساعة)، والنسخ مرتفعة من الببتيد الجينات المضادة للميكروبات (2-24 ساعة)، وتشكيل ندبة. أساسا كل نوع البرية الحيوانات الكبار البقاء على قيد الحياة جرح، حيثكما المسوخ المعيبة في إصلاح الجرح أو استجابات أخرى تظهر انخفاض البقاء على قيد الحياة. وتعرض بروتوكولات مفصلة لإبرة وجرح الليزر، وفحوصات لتحديد الكميات والتصور الردود الجروح والعمليات إصلاح (ديناميات الكالسيوم، وديناميات الأكتين، مضادات الميكروبات الببتيد الاستقراء، والبقاء على قيد الحياة).

سوف الليزر أو جرح إبرة يؤدي الارتفاع السريع والمستمر في البشرة مستويات الكالسيوم، كما تصور مع أجهزة استشعار الكالسيوم مثل GCaMPs (الشكل 1). يحدث ارتفاع الكالسيوم في غضون ثوان، ويبقى مرتفعا لعشرات من الدقائق. إبرة اصابة بتكاثر النتائج في تشكيل حلقات F-أكتين في مو...

Watch this Scientific Journal Video about Methods for Skin Wounding and Assays for Wound Responses in C. elegans at JoVE.com

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

طرق للبشرة إصابة وفحوصات لالردود الجرح في<em&gt; C. ايليجانس</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

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Biology

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

10.2K Views.  University of California, San Diego. The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity. 

Video: Methods for Skin Wounding and Assays for Wound Responses in C. elegans

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

نماذج لتوصيف الدوائية من المسوخ جيم ايليجانس نقل متشابك

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

فحوصات Neurocircuit عن المضبوطات في المسوخ من الصرع ذبابة الفاكهة</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

الأساسية<em&gt; Caenorhabditis ايليجانس</em&gt; طرق: التزامن والمراقبة

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

منهج في التثقيف الجنينية<em&gt; C. ايليجانس</em&gt; خلايا

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

نموذج نقص الأكسجين المجاعة لنقص التروية / ضخه في<em&gt; C. ايليجانس</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

طرق لتقييم التحت خلوية من المقصورات في العضلات<em&gt; C. ايليجانس</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

التحقيق في نشر وسمية البروتينات مثل بريون باستخدام Metazoan نموذج حي<em&gt; C. ايليجانس</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

تتبع وقياس العمليات التنموية في<em&gt; C. ايليجانس</em&gt; استخدام أدوات المصدر المفتوح

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

أساليب "دراسة التغيرات" في "تجميع البروتين الأصيل" مع التقدم في السن في ايليجانس كاينورهابديتيس

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

قياسات استجابات الإجهاد الفسيولوجي في C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...