The authors would like to thank Chiyoko Kobayashi and Kiyokazu Agata for helpful advice on planarian transplantation as well as past and present members of the S&aacute;nchez lab for invaluable discussions during the development of these techniques. This work was supported by NIH Training Grant (5T32 HD0791) to OCG and NIH R37GM057260 to ASA. ASA is a Howard Hughes Medical Institute Investigator.

Note: this protocol suggests the use of potentially hazardous materials (lead and chloretone). Acquire, read, and follow MSDS for all potentially hazardous materials.
1. Animal Culture, Selection, and Preparation
<ol>
 <li>For animal culture and handling use planarian water (1X Montju&iuml;c salts9) and plastic transfer pipettes.</li>
 <li>Sexual biotype Schmidtea mediterranea can be used when raised in the laboratory under normal culture conditions10. To produce asexual specimens of requisite size, S. mediterranea raised at room temperature under normal conditions9 should be fed at double to triple normal frequency (2-3 times per week) for one to two months prior to use. Alternatively, asexual animals fed at normal frequency may be kept at 10 degrees Celsius indefinitely in order to increase their average size.</li>
 <li>Select animals that are between 1 to 2 cm in length and wider than 2 mm then starve animals 3-7 days prior to use.</li>
 <li>If performing any pharmacological or radiological treatments on intended hosts, donors, or partially irradiated animals, perform treatment(s) at this point.</li>
 <li>If pharmacological treatments were performed that required feeding the animals, starve animals an additional 3 to 7 days prior to use.</li>
</ol>
2. Preparation of Solutions and Materials
<ol>
 <li>Prepare chloretone solution, a mild local anesthetic, by dissolving 0.1-0.2% w/v chloretone in planarian water and chilling the solution on ice.</li>
 <li>If performing partial irradiation only, proceed to step 2.7. For tissue transplantation continue on to step 2.3.</li>
 <li>Using a Bunsen burner, bend 0.75 mm interior diameter, used for cutting the graft tissue, and 0.7 mm exterior diameter, used for creating a hole in the host which will receive the graft, capillary tubes to a 90&deg; angle at 1-2 cm from the end of each tube. To save materials, bend both ends of each capillary tube and break them in half to produce two tools. Take care not to flame the very ends of the tubes.</li>
 <li>Cut the following papers to the indicated sizes:
 <ul>
 <li>Black filter paper (cut into rectangles approx. 2.5 cm x 1.5 cm)</li>
 <li>Whatman #3 filter paper (cut into rectangles approx. 2 cm x 0.5 cm)</li>
 <li>Kimwipe (folded and cut into wads approx. 3 cm x 0.5 cm x 4 ply)</li>
 <li>Cigarette rolling paper (remove gum strip and cut into rectangles approx. 3 cm x 2 cm)</li>
 </ul>
 </li>
 <li>Prepare modified Holtfreter's solution (3.5 g/L NaCl, 0.2 g/L NaHCO3, 0.05 g/L KCl, 0.2 g/L MgSO4, 0.1 g/L CaCl2, pH 7.0-7.5) and casein saturated Holtfreter's solution and chill both to 4 &deg;C.</li>
 <li>Attach a folded Kimwipe to a square of Parafilm and place on Peltier cooler plate or other cooling device situated under a dissecting microscope. Saturate the Kimwipe with chilled Holtfreter's solution and place two black filter paper rectangles on the Kimwipe.</li>
 <li>Line Petri dishes with Whatman #2 filter paper. Moisten the filter paper with Holtfreter's solution and chill the dishes on ice. For partial irradiation a larger dish and filter paper liner may be used.</li>
</ol>
3. Anesthetization and Immobilization
<ol>
 <li>Fill a Petri dish with chilled chloretone solution and pipette worms into the dish. For transplantation only anesthetize one host and one donor at a time. For partial irradiation many (n &gt; 10) animals can be anesthetized at once.</li>
 <li>Allow worms to soak in the chloretone solution until they become motionless (5-10 min).</li>
 <li>Rinse worms by pipetting them into a dish filled with chilled Holtfreter's solution.</li>
 <li>Immobilize animals by pipetting them onto black filter paper saturated with chilled Holtfreter's solution and orient them ventral side down with forceps. If animals are able to locomote, soak up excess Holtfreter's solution, slightly decrease the temperature of your Peltier or cold plate, or increase the length of chloretone treatment.</li>
</ol>
4. Partial Irradiation
Note: Follow these steps to prepare animals for partial irradiation. If performing transplantation instead, proceed to section 5.
<ol>
 <li>Place a chilled Petri dish from step 2.7 on ice in an ice bucket that will fit inside a top source X-ray irradiator.</li>
 <li>Arrange anesthetized animals in a Petri dish by moving the black filter paper on which they are immobilized. Using forceps to move anesthetized worms directly may injure them.</li>
 <li>Transport arranged animals to a top-source X-ray irradiator and situate the ice bucket such that the distance from the cathode tube to the animals is minimized, thus maximizing the effective dose rate.</li>
 <li>Position lead shield(s) (Figure 1) between the animals and the cathode tube as desired. Shields should be 4.5 to 6 mm thick to allow for 97 to 99% attenuation of a 325kV X-ray beam11.</li>
 <li>Deliver X-ray dose. If complete stem cell ablation from non-shielded regions is desired, deliver 30 Gy or greater using an X-ray irradiator. For reference, 30 Gy is equivalent to 3.6 minutes at 320 kilovolts and 10 milliamps in a Precision X-Ray Inc. XRAD320 with a field-to-source distance of 30 centimeters.</li>
 <li>Immediately after dosing is complete, handling worms by the black filter paper, transfer animals into chilled planarian water. Allow the planarian water to warm to room temperature and the animals to dislodge themselves from the black filter paper. The partial irradiation procedure is now complete.</li>
</ol>
5. Tissue Transplantation
<ol>
 <li>Using a transfer pipette and forceps, arrange anesthetized host and donor worms on separate rectangles of black filter paper on the Kimwipe which has been cooled on the Peltier or cooler plate under the dissecting microscope.</li>
 <li>Using a 0.75 mm inner diameter capillary tube cut out the graft plug from the donor and, using forceps, place it on an out of the way portion of the host. If graft material gets stuck in the capillary tube, dislodge with forceps.</li>
 <li>Using a 0.7 mm outer diameter capillary tube remove a plug from the host and using forceps position the graft into the hole that is left behind.</li>
 <li>Transfer the transplanted host on its black filter paper rectangle into the Petri dish prepared in step 2.7.</li>
 <li>Wet a piece of rolling paper with casein saturated Holtfreter's solution and place it on top of the transplanted host as diagramed in Figure 2A.</li>
 <li>Soak four pieces of filter paper in casein saturated Holtfreter's solution and encase the transplanted host as diagramed in Figure 2B.</li>
 <li>Soak four wads of cut Kimwipe in casein saturated Holtfreter's solution and lay them over the filter paper from step 5.6 (Figure 2B). Replace the lid and put the Petri dish on ice.</li>
 <li>Transfer the donor worm into planarian water to recover, heal, and regenerate.</li>
 <li>When all transplants are completed, place the transplanted worms into a 10 &deg;C incubator overnight.</li>
 <li>The following morning, taking care not to disturb the graft, uncover the worm and transfer it (on its black filter paper) to a Petri dish filled with planaria water.</li>
 <li>Either allow the worm to dislodge itself from the filter paper or gently remove it with forceps.</li>
 <li>Change the planarian water once every 2-3 days.</li>
</ol>
6. Representative Results
Immediately following partial irradiation planaria will appear normal and unaffected. Depending on the delivered dose and the geometry of the shield used, irradiated tissue may regress and even disintegrate7. Shielded tissue should remain intact. Following tissue regression and a loss of tissue integrity, a blastema will form and missing structures will be regenerated (Figure 3A). If an amputation is made in the partially irradiated region, the irradiated tissue will be rescued (i.e. prevented from regressing or disintegrating) (Figure 3B). In both the uninjured and the amputated case regeneration will be delayed as compared to an amputated non-irradiated planaria (Figure 3C). If an X-ray dose of 30 Gy was delivered and the partially irradiated animal was uninjured, successful stem cell ablation in a pattern corresponding with the lead shield used can be confirmed 2 to 3 days following partial irradiation by in situ hybridization for the stem cell marker Smed-piwi-112 (a.k.a. smedwi-1) (Figure 4).
The morning following transplantation, a successful graft of the transplanted tissue should be obvious within the host tissue, having adhered to both the dorsal and ventral surfaces of the host (Figure 5A). Occasionally, the graft will adhere to only the ventral or dorsal surface. If the transplantation was completely unsuccessful, no sign of the graft will be visible from either the dorsal or ventral surface of the host (Figure 5B). Shortly following a successful graft of non-irradiated tissue into a host that had been ablated of stem cells by lethal irradiation13,14 , in situ hybridization for Smed-piwi-1 will reveal that stem cells are present primarily within the graft (Figure 5C). Additionally, successful grafts of non-irradiated tissue into lethally irradiated hosts will result in rescue of host tissue and long-term survival of the host15.
<img alt="Figure 1" src="/files/ftp_upload/4015/4015fig1.jpg" /> 
Figure 1. General arrangement of basic components for partial irradiation. In an X-ray irradiator with a top positioned X-ray source (cathode tube) the anesthetized planarian is positioned directly below the X-ray source within the irradiation field. In order to maximize the X-ray dose rate, the distance between the planarian and the X-ray source should be minimized. A lead shield should be positioned between the cathode tube and the anesthetized worm, as close to the worm as practical. The lead shield should be designed, manufactured, and positioned so that it shields the desired tissue but completely exposes the rest of the worm. Many commercial manufactures will produce custom lead shields from your simple design diagram; we have successfully used Alpha Systems Corp. (Bluffdale, UT). The lead should be sufficiently thick to allow for the desired amount of shielding. For example, if near complete shielding of a 320 kV X-ray beam is desired, the lead should be 4.5 to 6 mm thick. The planarian and shield are positioned on a Holtfreter's soaked filter paper lined Petri dish which rests in an ice bucket. Given a sufficiently large X-ray irradiation field and a number of identical lead shields, many specimens may be partially irradiated at one time (not pictured).
<img alt="Figure 2" src="/files/ftp_upload/4015/4015fig2.jpg" /> 
Figure 2. Building the recovery chamber. (A) An exploded view of the tissue transplantation recovery chamber, showing all of the components that are layered on top of one another after being soaked in casein saturated Holtfreter's Solution. (B) A nearly completed recovery chamber, illustrating the interlocking placement of the Whatman #3 filter paper rectangles which snugly encase the anesthetized planarian, preventing movement during healing. The careful construction of the recovery chamber prevents animal movement and desiccation, promoting rapid healing and greater efficacy of tissue transplantation.
<img alt="Figure 3" src="/files/ftp_upload/4015/4015fig3.jpg" /> 
Figure 3. Representative outcomes of simple posterior partial irradiation. As Dubois described7, (A) when the posterior half of planarians were shielded with lead and then exposed to x-ray irradiation the anterior tissue was observed to regress back to the boundary between the irradiated and shielded tissue at which point unpigmented blastemas formed and the animals began to regenerate. (B) On the other hand, when animals were decapitated following the same partial irradiation performed in (A), tissue regression was not observed and the remaining irradiated anterior tissue was rescued. The decapitated partially irradiated animals regenerated heads (B), however, regeneration was significantly delayed as compared to unirradiated decapitated controls (C).
<img alt="Figure 4" src="/files/ftp_upload/4015/4015fig4.jpg" /> 
Figure 4. Representative outcome of partial stem cell ablation following partial irradiation. Whole-mount In situ hybridization (WISH) for the stem cell marker Smed-piwi-1 reveals that wild type planaria have stem cells distributed throughout their bodies with the exception of the tissue anterior to the photoreceptors (arrowhead) and the pharynx proper (asterisk)14. (A) WISH for Smed-piwi-1 in irradiated but fully shielded control planaria fixed three days following irradiation shows a stem cell distribution that is indistinguishable from that of wild type planaria. (B) On the other hand, WISH for Smed-piwi-1 in animals that were only partially shielded, leaving the anterior and posterior exposed, but were also fixed three days following irradiation shows that stem cells are ablated from the non-shielded regions. Scale bars are 500 microns.
<img alt="Figure 5" src="/files/ftp_upload/4015/4015fig5.jpg" /> 
Figure 5. Examples of successful and unsuccessful tissue transplantation. (A) Live images of dorsal and ventral views of a successfully transplanted planarian three days following transplantation. The graft (indicated) is clearly visible on both dorsal and ventral surfaces and is surrounded with characteristic unpigmented tissue at the graft-host interface. (B) Correspondingly, unsuccessful transplantations display no visible graft tissue at the transplantation site (indicated) and instead show a healed, unpigmented, lateral wound from the failed transplantation. A graft that adheres to only the dorsal or the ventral surface can resemble a successful transplantation (A) when viewed from one side and an unsuccessful transplantation (B) when viewed from the other. (C) When wild type (wt) tissue is grafted into an irradiated host that has been ablated of resident stem cells and the transplanted stem cells are later revealed by WISH for Smed-piwi-1 two days following transplantation, the success of the transplantation is clearly displayed by the specific presence of stem cells only in or around the graft location (arrowheads). Scale bars are 500 microns.

<ol>
	<li>Morgan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+the+regeneration+of+Planaria+maculata.">Experimental studies of the regeneration of Planaria maculata.</a> Arch. Entw. Mech. Org. 7, 364-397 .</li><li>Reddien, P. W., Sanchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+planarian+regeneration.">Fundamentals of planarian regeneration.</a> Annu Rev Cell Dev Biol. 20, 725-757 (2004).</li><li>Randolph, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regeneration+of+the+tail+in+lumbriculus.">The regeneration of the tail in lumbriculus.</a> J. Morphol. 7, 317-344 .</li><li>Wagner, D. E., Wang, I. E., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+neoblasts+are+pluripotent+adult+stem+cells+that+underlie+planarian+regeneration.">Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration.</a> Science. 332, 811-816 (2011).</li><li>Santos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transplantation+in+planaria.">Studies on transplantation in planaria.</a> Biological Bulletin. 57, 188-197 (1929).</li><li>Morgan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+grafted+pieces+of+planarians.">Regeneration of grafted pieces of planarians.</a> J. Exp. Zo&#246;l. 3, 269-294 (1906).</li><li>Dubois, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+&#225;+l+'&#232;tude+de+la+migration+des+cellules+de+règènèration+chez+les+Planaires+dulcicoles.">Contribution &#225; l '&#232;tude de la migration des cellules de règènèration chez les Planaires dulcicoles.</a> Bull. Biol. Fr. Belg. 83, 213-283 (1949).</li><li>Purton, L. E., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+factors+in+murine+hematopoietic+stem+cell+assays.">Limiting factors in murine hematopoietic stem cell assays.</a> Cell Stem Cell. 1, 263-270 (2007).</li><li>Cebria, F., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+homologs+of+netrin+and+netrin+receptor+are+required+for+proper+regeneration+of+the+central+nervous+system+and+the+maintenance+of+nervous+system+architecture.">Planarian homologs of netrin and netrin receptor are required for proper regeneration of the central nervous system and the maintenance of nervous system architecture.</a> Development. , 132-3691 (2005).</li><li>Newmark, P. A., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+specifically+labels+the+regenerative+stem+cells+of+planarians.">Bromodeoxyuridine specifically labels the regenerative stem cells of planarians.</a> Dev. Biol. 220, 142-153 (2000).</li><li>Miller, W., Kennedy, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+attenuation+in+lead,+aluminum,+and+concrete+in+the+range+275+to+525+kilovolts.">X-ray attenuation in lead, aluminum, and concrete in the range 275 to 525 kilovolts.</a> Radiology. 65, 920-925 (1955).</li><li>Pearson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-based+whole-mount+in+situ+hybridization+method+for+planarians.">Formaldehyde-based whole-mount in situ hybridization method for planarians.</a> Dev. Dyn. 238, 443-450 (2009).</li><li>Hayashi, T., Asami, M., Higuchi, S., Shibata, N., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+planarian+X-ray-sensitive+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting.</a> Dev. Growth Differ. 48, 371-380 (2006).</li><li>Reddien, P. W., Oviedo, N. J., Jennings, J. R., Jenkin, J. C., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMEDWI-2+is+a+PIWI-like+protein+that+regulates+planarian+stem+cells.">SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells.</a> Science. 310, 1327-1330 (2005).</li><li>St&#233;phan-Dubois, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Les+cellules+de+r&#233;g&#233;n&#233;ration+chez+la+planaire+Dendrocoleum+lacteum.">Les cellules de r&#233;g&#233;n&#233;ration chez la planaire Dendrocoleum lacteum.</a> Bulletin de la Société Zooologique de France. 86, 172-185 (1961).</li><li>Gurley, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+secreted+Wnt+pathway+components+reveals+unexpected+complexity+of+the+planarian+amputation+response.">Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response.</a> Dev. Biol. 347, 24-39 (2010).</li><li>Salvetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+stem+cell+plasticity:+neoblast+repopulation+in+non-lethally+irradiated+planarians.">Adult stem cell plasticity: neoblast repopulation in non-lethally irradiated planarians.</a> Dev. Biol. 328, 305-314 (2009).</li></ol>

No conflicts of interest declared.

Importance of immobilization
Immobilization is by far the most critical step for the successful completion of any of these processes. If planaria are improperly immobilized prior to partial irradiation, they may move beneath the lead shield, producing inconsistent and confounding results. Additionally, if animals are insufficiently immobilized following transplantation, the host worm will likely move away from the graft tissue, resulting in a failure of the graft to heal properly to the host. Pro...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>General Purpose Transfer Pipette</td>
 <td>Samco</td>
 <td>691</td>
 </tr>
 <tr>
 <td>Capillary tubes (ID 0.75 mm)</td>
 <td>FHC</td>
 <td>30-30-0</td>
 </tr>
 <tr>
 <td>Capillary tubes (OD 0.7 mm)</td>
 <td>FHC</td>
 <td>30-50-08</td>
 </tr>
 <tr>
 <td>Parafilm M</td>
 <td>VWR</td>
 <td>52858-076</td>
 </tr>
 <tr>
 <td>Kimwipes 34155</td>
 <td>VWR</td>
 <td>500029-891</td>
 </tr>
 <tr>
 <td>Black filter paper</td>
 <td>Schleicher &amp; Schuell</td>
 <td>10310809</td>
 </tr>
 <tr>
 <td>Whatman #2 filter paper 1002-055</td>
 <td>Fisher Scientific</td>
 <td>09-810B</td>
 </tr>
 <tr>
 <td>Whatman #3 filter paper 1003-185</td>
 <td>Fisher Scientific</td>
 <td>09-820E</td>
 </tr>
 <tr>
 <td>Cigarette rolling paper</td>
 <td>Zig-Zag, original</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>Petri dishes</td>
 <td>VWR</td>
 <td>82050-544</td>
 </tr>
 <tr>
 <td>Forceps DUMONT, INOX #5</td>
 <td>FST</td>
 <td>11251-20</td>
 </tr>
 <tr>
 <td>Chloretone</td>
 <td>Sigma Aldrich</td>
 <td>112054</td>
 </tr>
 <tr>
 <td>Casein</td>
 <td>Sigma Aldrich</td>
 <td>C3400</td>
 </tr>
 <tr>
 <td>Lead Shields</td>
 <td>Alpha Systems Corp., Bluffdale, UT</td>
 <td>Custom design</td>
 </tr>
 <tr>
 <td>XRAD-320 Biological Irradiator</td>
 <td>Precision X-Ray, North Branford, CT</td>
 <td>NA</td>
 </tr>
 </tbody>
</table>

planarian immobilization, partial irradiation, and tissue transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

Watch this Scientific Journal Video about Planarian Immobilization, Partial Irradiation, and Tissue Transplantation at JoVE.com

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

planarian-immobilization-partial-irradiation-tissue

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19.4K Views.  University of Utah School of Medicine. An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Video: Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.