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).

The following protocols describe the detailed procedure for C. elegans skin wounding and for assaying wound responses.
1. Needle Wounding3,4
<ol>
	<li>Grow healthy unstarved worms on standard NGM (nematode growth medium) plates with E. coli OP50 bacteria as food, maintained in a 20 &#176;C incubator. 
	NOTE: Methods for NGM agar plates and routine cultivation of C. elegans can be found at www.wormbook.org6.</li>
	<li>Pick 25 L4 stage worms to a freshly seeded plate 1 day before wounding and culture at 20 &#176;C O/N.</li>
	<li>Before wounding, pull needles from capillaries using a needle puller. The needles used in wounding are identical to those used for microinjection of C. elegans.</li>
	<li>Ensure that all the worms are at young adult stage. Place the plate of worms on ice for about 30 min. Cooling causes the animals to be sluggish, facilitating needle wounding. 
	NOTE: Within 5 min of removal from the ice, the worms will start to move normally. It is possible but challenging to wound moving worms. With experience, 5 min is enough time to wound 25 worms. Immobilize with drugs such as levamisole (as used in laser wounding, see below), although animals will take longer to recover.</li>
	<li>Remove the agar plate from the ice to a dissecting stereomicroscope. Using a worm pick, move 25 worms into the center of the agar plate.</li>
	<li>Place the needle into a 100 &#181;l pipette tip to more easily hold the needle. Puncture the anterior or posterior part of the body of the worm with the needle, avoiding the gonad. In general, attempt to wound between the posterior pharynx and anterior gonad, or between the posterior gonad and the rectum. Re-use needles many times, until they break. 
	NOTE: Ensure the wounds are brief, gentle pricks that puncture the cuticle and skin, penetrating ~5-10 &#181;m into the animal. The needle should enter the animal at approximately a right angle to the skin. The wounds should not cause obvious damage to internal organs, or immediate rupture of the body. Observe a small amount of cytoplasm oozing out for a few seconds after the wound; however, if cellular contents leak out over a prolonged time the animals will not survive. For optimal imaging, wound the lateral epidermal syncytium (hyp7) or the lateral seam. 
	NOTE: This procedure includes the use of a glass needle.</li>
	<li>Allow worms to recover at RT&#160;then culture at 20 &#176;C. Judge the success of needle wounding by a number of assays described below. Greater than 95% of wild type animals survive 24 hr after needle wounding in the young adult stage. The effect of wounding in larvae or older adults has not yet been extensively analyzed.</li>
</ol>
2. Laser Wounding3
Use femtosecond laser irradiation (800 nm) to perform more precise wounding.
<ol>
	<li>Prepare agar pads on a glass slide with melted 2% agar7. Ensure the pads are similar to the agar pads used for live imaging of C. elegans.</li>
	<li>Transfer 10 young adult worms onto the agarose pad with worm pick, and add a 2 &#181;l drop of 12 mM Levamisole solution. Cover the animals and the liquid with a cover slip. Wait 1-2 min for the worms to paralyze.</li>
	<li>Place the glass slide onto a spinning disk confocal microscope. Move the stage to find the worms and focus on the lateral anterior or posterior syncytial epidermis with a 100X&#160;objective (NA 1.4-1.46).</li>
	<li>Set the power of the femtosecond laser to 140 mW (measured before the objective). Use the femtosecond laser at a repetition rate of 80 MHz. 
	NOTE: Two pulses of 200 msec each (separated by 20 msec) are sufficient for wounding in our hands.</li>
	<li>Focus on the apical surface of the epidermal cell and wound the epidermis. Observe local disruption of the cytoplasm (bubbling), or local bleaching of any fluorescent marker used. 
	NOTE: Follow appropriate laser safety procedures when using the femtosecond laser. Line or point scans using femtosecond lasers such as those in two photon microscopes should provide sufficient power for laser wounding. 
	NOTE: While we do not have direct experience of using other lasers, in principle wounding should be possible using conventional UV lasers (as used in C. elegans cell ablations). Optionally, use a Fluorescence Recovery After Photobleaching (FRAP) module on spinning disk confocal to wound the epidermis (N. Pujol, personal communication).</li>
</ol>
3. Visualization of Epidermal Ca2+&#160;Responses to Wounding
<ol>
	<li>Use transgenic C. elegans strains that express Ca2+ sensors (such as those of the GCaMP series) in the epidermis, under the control of the adult epidermal specific promoter col-19 (Figure 1A; transgenes are listed in Table 1). Use a transgene expressing a red fluorescent protein such as tdTomato as an internal control for transgene expression level. 
	NOTE: We have used tdTomato as an internal control for transgene expression as epidermal tdTomato fluorescence is relatively stable and does not interfere with GCaMP imaging.</li>
	<li>Both needle and laser wounding trigger Ca2+ elevation in the epidermis. However, as needle wounding cannot be readily performed on a spinning disk confocal, laser wounding is preferable for quantitative analysis of the Ca2+ response (Figure 1).</li>
	<li>Acquire time lapse images in multi-dimensional acquisition mode (interval time 2 sec with 114 msec excitation laser exposure) using a spinning disk confocal with 100X&#160;objective (NA 1.4-1.46) and appropriate filter sets (GFP filters will work for GCaMP3). 
	NOTE: Acquire time-lapse images every 30 sec for about 2 hr&#160;to examine the Ca2+ response to wounding over a longer time course.</li>
	<li>Using image analysis software measure the average GCaMP fluorescence in ten equivalent regions of interest (ROI), five of which are centered on the epidermal cell cytosol and five in the background.</li>
	<li>Obtain baseline fluorescence (F0) by averaging fluorescence in 5 ROIs in the epidermis then subtracting the average of 5 ROIs in the background before injury. Express the change in fluorescence &#916;F as the ratio of change with respect to the baseline [(Ft-F0)/F0].</li>
</ol>
4. Visualization of F-actin Dynamics After Wounding
<ol>
	<li>NOTE: In response to needle wounding, observe an actin ring at the wound site that gradually closes around the wound. This protocol section assays wound closure by measuring the actin ring diameter.</li>
	<li>Generate transgenic worms that express an F-actin marker such as the Drosophila moesin actin binding domain fused to GFP, expressed under the control of an epidermal specific promoter such as col-19 (Figure 2A) (transgene juIs352). 
	NOTE: We have obtained similar results using other actin binding domain markers such as Lifeact or F-tractin (unpublished results).</li>
	<li>Perform needle wounding on Pcol-19-GFP-moesin transgenic worms. After needle wounding, observe a ring of GFP-moesin around the wound site by ~5 min. The actin ring becomes smaller in diameter and eventually closes 2-3 hr post wounding in wild type animals (Figure 2A).</li>
	<li>Prepare a 2% agar pad on a glass slide and transfer 10 worms onto the pad in 2 &#181;l 12 mM Levamisole solution. Image the GFP-moesin rings 1 hr after wounding using conventional confocal microscopy. 
	NOTE: Use confocal microscopy to acquire z-stacks (13 x 0.5 &#181;m) of actin rings 1 hr after wounding.</li>
	<li>Measure the actin ring diameter after wounding. To quantitate epidermal GFP-moesin, first make a maximum intensity projection of the z-stack and then draw 4 line scans (at 45&#176; to each other) over the GFP-moesin ring. Ring diameter is defined as the average peak to peak distance of the four line scans (Figure 2B). For rings that have already closed, the diameter is defined as 0 &#181;m. 
	NOTE: F-actin rings are conveniently measured 1 hr post wounding as this is approximately half way through the wound closure process in the wild type. F-actin rings can also be measured at multiple time points to derive a time course of wound closure. Time lapse movies of wound closure can be acquired using levamisole-immobilized worms and spinning disk confocal microscopy.</li>
</ol>
5. Assay Induction of Epidermal Antimicrobial Peptides

NOTE: Wounding also triggers innate immune responses in the C. elegans skin. Transcription of genes encoding antimicrobial peptides (AMPs) is induced in the epidermis. The nlp-29 (neuropeptide-like protein) AMP is strongly induced by wounding4. To assay how the epidermis controls AMP expression after wounding, use transgenic reporters or real-time PCR to detect individual AMP transcript levels.
<ol>
	<li>Transgenic reporter assay for innate immune response to wounding4.</li>
	<li>Perform needle wounding (as in protocol 1) on adult animals expressing the transgenic reporter frIs7, which contains Pnlp-29-GFPand Pcol-12-DsRed as an internal control. Observe unwounded adult animals as dark red or orange in a GFP long pass filter. 
	NOTE: Wounding wild-type worms will induce Pnlp-29-GFP expression but will not affect Pcol-12-dsRed, resulting in worms that appear orange, yellow, or green under GFP long pass illumination. Loss of function of genes in the p38 MAPK pathway, such as pmk-1 or nsy-1 will block nlp-29-GFP induction after wounding4. Pnlp-29-GFP is expressed at high levels in larval stages</li>
	<li>Perform AMP induction assays in young adult animals. Ensure that unwounded control animals have low levels of GFP. 
	NOTE:Pnlp-29-GFP expression is also induced by osmotic stress8, and may be sensitive to other environmental stresses.</li>
	<li>6 hr after wounding, score the numbers of worms that have Pnlp-29-GFP induction (Figure 3)9 using a fluorescence dissecting scope with GFP long-pass filter. 
	NOTE: At 1 hr post-wounding Pnlp-29-GFP is visibly induced, reaching a peak about 6 hr post-wounding and then remaining elevated up to 24 hr post wounding. Quantify expression by visual scoring4,9. Alternatively, quantify expression levels using a fluorescence activated worm sorter4. For the worm sorter, at least 50 animals need to be wounded.</li>
	<li>Real-time PCR assay for quantitating transcript levels of the individual AMP genes. Assay transcript levels for several genes: nlp-29, nlp-30, cnc-1, and cnc-5.</li>
	<li>Perform needle wounding on wild-type or mutant worms (see protocol 1)</li>
	<li>Collect 50 wounded worms and 50 unwounded worms at the desired times (e.g. 3, 6, 24 hr) post-wounding.</li>
	<li>Extract RNA using a commercial kit following the manufacturer&#8217;s instructions.</li>
	<li>Perform reverse transcription to get total 20 &#181;l cDNA using a commercial reverse transcriptase kit.</li>
	<li>For RT-PCR, use 0.2 &#181;l cDNA (1/40) from each sample in combination with a green supermix with fluorescein and 0.2 &#181;M primers. To compare RNA quality, use ama-1 or snb-1 RT-PCR as reference; compare the AMP relative expression level by normalizing to internal control (ama-1 or snb-1), or compare fold induction post-wounding (AMP induction in wounded worms/unwounded worms) by normalizing to internal control. 
	NOTE: cnc-1 and cnc-5 are caenacin family antimicrobial peptides whose transcription is activated by wounding10. Typically, needle wounding induces expression (~500 fold for cnc-1) over a time course of 4 hr10. Primers used in published work are listed in Table 2.</li>
</ol>
6. Assay Survival After Wounding
<ol>
	<li>NOTE: Wounding activates at least two independent signaling pathways in the epidermis: the Ca2+ dependent wound repair pathway and the innate immune response pathway. Both pathways are required for full survival of sterile wounding. To test whether a mutant or RNAi treatment affects wound healing, measure survival at 24-48 hr post wounding. Optionally, use conventional lifespan assays to determine the effect of wounding on longevity4,9.</li>
	<li>Perform needle wounding on young adult (L4 + 24 hr) worms (50 worms, repeat several times) following protocol 1 above. Maintain an equivalent number of unwounded worms as controls.</li>
	<li>Check survival every 24 hr (transfer wounded worms to a new agar plate every 24 hr). Observe ~50% survival 24 hr after wounding in mutants that are defective in wound repair such as gtl-2 or tir-1, when normalized to unwounded control animals. Death is defined as a lack of locomotor response to touch by worm pick.</li>
</ol>

<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. D., Xu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+epidermis+as+a+model+skin.+II:+differentiation+and+physiological+roles.">The Caenorhabditis elegans epidermis as a model skin. II: differentiation and physiological roles.</a> Wiley Interdiscip Rev Dev Biol. 1, 879-902 (2012).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook : the online review of C. elegans biology. , 1-11 .</li><li>Bargmann, C. I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+killing+of+cells+in+Caenorhabditis+elegans.+Methods+in+cell+biology.">Laser killing of cells in Caenorhabditis elegans. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroimmune+regulation+of+antimicrobial+peptide+expression+by+a+noncanonical+TGF-beta+signaling+pathway+in+Caenorhabditis+elegans+epidermis.">Neuroimmune regulation of antimicrobial peptide expression by a noncanonical TGF-beta signaling pathway in Caenorhabditis elegans epidermis.</a> Nat Immunol. 10, 249-256 (2009).</li><li>Zhao, X., 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=Microfluidic+chip-based+C.+elegans+microinjection+system+for+investigating+cell-cell+communication+in+vivo.">Microfluidic chip-based C. elegans microinjection system for investigating cell-cell communication in vivo.</a> Biosensor., &amp; Bioelectronics. 50, 28-34 (2013).</li><li>Jansson, H. 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.

The methods presented here for needle and laser wounding provide complementary approaches for assessing the ability of the epidermal epithelium to repair damage. Laser wounding is relatively localized and (depending on laser configuration) can be confined to the epidermis, whereas needle wounding disrupts the epidermis, cuticle, and likely internal basement membranes. Needle wounding may more accurately resemble wounds inflicted by pathogens or mechanical damage in the natural environment. As a rule, needle wounding requ...

Skin wound healing mechanisms are of basic biological interest and relevant to human health. Wound healing in vertebrates and mammals comprises a complex series of coordinated responses of multiple tissues and signaling1. Many simple genetic model organisms are also capable of healing skin wounds2. It is therefore of interest to analyze genetically tractable models of skin wound repair. We and others have begun to use Caenorhabditis elegans as new model for skin wound healing3,4. The goal of this protocol is to enable a broader set of researchers to use C. elegans as a tool to investigate molecular and cellular mechanisms of epidermal wound healing.
The C. elegans skin comprises the epidermis (also known as hypodermis) and the extracellular cuticle5. The adult epidermis is formed from a small number of multinucleate syncytia, of which the largest is the syncytium known as hyp7. The epidermis is a simple epithelium that secretes the cuticle on its apical surface. The skin can actively defend against skin-penetrating pathogens and repair small wounds4. Wound repair of the C. elegans skin is robust, as almost all wild type animals survive can survive small puncture wounds caused by needles, or local skin damage caused by laser irradiation. C. elegans skin wounding triggers a suite of responses, including an epidermal innate immune response, wound closure, and scar formation4. The adult epidermis is post-mitotic, and wound healing involves local cellular responses as opposed to epidermal proliferation or cell migration. We have shown that skin wounding triggers a large and sustained increase in epidermal Ca2+, requiring the membrane TRPM channel GTL-2 and internal Ca2+ stores3. The epidermal Ca2+ signal is required for formation and closure of F-actin rings at the wound site. Wounding also induces innate immune responses that activate the transcription of AMPs such as nlp-29. The wounding-induced transcription of AMPs is dependent on a TIR-1/PMK-1 p38 MAP kinase cascade acting autonomously in the epidermis4. Defects in either the Ca2+ signaling pathway or in the innate immune response will lead to lower survival post-wounding. The relatively simple structure of the C. elegans epidermis, its genetic tractability and advantages for in vivo imaging make it an excellent system to study multiple aspects of wound repair.
Here we present protocols for the two common methods of wounding: needle wounding and laser wounding. Needle wounding requires no specialized equipment (other than the needle puller), and with experience can be performed on hundreds of worms per day. Needle wounding is performed on animals growing on agar plates. In contrast, laser wounding is performed on anesthetized animals mounted on agar pads under a coverslip, and is suited for live imaging of the cellular responses to damage.

<table border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="22">
			<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

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Laser or needle wounding will trigger rapid and sustained elevation in epidermal Ca levels, as visualized with Ca sensors such as GCaMPs (Figure 1). The Ca elevation occurs within seconds, and remains elevated for tens of minutes. Needle wounding reproducibly results in the formation of F-actin rings at wound sites (Figure 2); these appear within minutes, and gradually close over 1-2 hr&#160;after wounding. Actin rings are less often formed after more precise laser wounding. Both needle ...

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.

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

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

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

Research

JoVE Journal

Biology

10.2K 조회수.  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. 

비디오: 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.

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.

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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.

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.

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.

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.

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 ...

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.

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.

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.

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