This work was supported by NIH grant&#160;R01 GM090305 (WFM).&#160;The procedure described here was developed at the Physiology Course of the Marine Biological Laboratory during the summer of 2012, and we thank the students, instructors, and course directors for many helpful and stimulating discussions.

1. Cell Culture and Collection
<ol>
	<li>Obtain Stentor coeruleus cells either from natural ponds as described by Tartar7 or else from a commercial supplier (see Reagents).</li>
	<li>Maintain cultures by growing in the dark at 20 &#186;C in Modified Stentor Medium (MSM), which is adapted from recipes described by Tartar7 and De Terra12 containing 0.75 mM Na2CO3, 0.15 mM KHCO3, 0.15 mM NaNO3, 0.15 mM KH2PO4, 0.15 mM MgSO4, 0.5 mM CaCl2, and 1.47 mM NaCl.</li>
	<li>Supplement the MSM medium with a carbon source by adding living prey to the medium. Grow cultures of Chlamydomonas reinhardtii TAP medium using standard culture methods13. Prior to feeding, wash Chlamydomonas cells in MSM. Feed 300 ml&#160;Stentor cultures ~3 x 107 Chlamydomonas cells 2-3x per&#160;week.</li>
	<li>Collect Stentor cells for surgery using a pipette. Stentor tend to anchor to the bottom and sides of the culture dish and so, in order to collect cells, agitate the culture by bubbling air through a pipette tip until cells begin to detach and swim freely.</li>
	<li>Capture swimming cells using a pipette equipped with a wide-bore tip to avoid shearing the cells.</li>
	<li>Wash collected cells in fresh MSM.</li>
</ol>
2. Preparation of Tools for Microsurgery
<ol>
	<li>Hold two glass stirring rods over a Bunsen burner or other flame until the ends begin to melt.</li>
	<li>Touch the ends of the two rods together so the molten glass fuses.</li>
	<li>After creating a small droplet of molten glass between the two rods, remove them from the flame, allow to cool for 1-2 sec, and then pull them apart to make a clean break. Ideally, the glass will pull out into thin strings and break smoothly as the glass cools resulting in a needle that is approximately 1-2 cm in length with fine point at the end (Figure 2).</li>
	<li>If the resulting needle is suboptimal the tips may be broken into glass waste and the rods may be reused.</li>
</ol>
3. Mounting Cells for Microsurgery and Observation (Normal Surgery)
<ol>
	<li>Prepare a square Vaseline walled chamber using a square rubber stamp coated in Vaseline and pressed onto a glass coverslip.</li>
	<li>Add MSM containing 2% Methyl Cellulose to the Vaseline square.</li>
	<li>Place the coverslip in a plastic adaptor, which allows the coverslip to be placed onto a microscope with a standard slide holder (Figure 3).</li>
	<li>Add washed cells from step 1.6 to the MSM plus methyl cellulose to impede the motility of the Stentor cells so that they stay in place during surgery.</li>
	<li>Arrange cells in the methyl cellulose so that they are evenly spaced and are pushed close enough to the surface of the coverslip to be in focus during imaging.</li>
</ol>
4. High Resolution Imaging of Wound Healing in Live Stentor
To simultaneously image a Stentor cell at high resolution while cutting it surgically under low magnification, construct a &#34;double decker&#34; microscope with a dissecting scope attached to the eyepiece head of the inverted scope so that it is focused on the top of the slide while the inverted scope is focused from beneath the slide.
<ol>
	<li>Put a slide on the inverted scope (use either a Zeiss Axiovert 200M or an Olympus IX80).</li>
	<li>Remove dissecting microscope body from its stand and determine approximate position above inverted microscope eyepiece for placing dissecting scope to allow the slide to be visualized at low magnification.</li>
	<li>Modify a cardboard box to have the same thickness as the desired spacing between inverted scope eyepiece head and dissecting scope, then tape into inverted scope using lab tape which can be easily removed when experiment is completed. Adjust the thickness of the box according to the exact configuration of the eyepiece head of the inverted microscope and on the size of the dissecting microscope so that the sample will be in the field of view without needing to move the dissecting scope after it is attached.</li>
	<li>Holding dissecting scope against box, move it until the slide is in focus, then tape onto the box again using lab tape. The final configuration is shown in Figure 4.</li>
	<li>Image cells through inverted scope using DIC optics and a 20X air objective lens. Use a long working distance DIC condenser to provide enough space to perform the microsurgery from above.</li>
	<li>Acquire images at a frame rate of 2 images/sec&#160;using a 512 x 512 CCD camera.</li>
	<li>Begin acquisition before starting to cut the cells so as to capture the initial cytoplasmic flow pattern and observe all stages of the wounding and wound healing process.</li>
	<li>Terminate acquisition when cell has resumed normal appearance and/or active motility, in order to visualize and measure cytoplasmic movements throughout the regeneration process.</li>
</ol>
5. Analyzing Cytoplasmic Flow During Wound Healing
Measure cytoplasmic flow using particle image velocimetry (PIV). This technique is routine in the study of fluid dynamics, and can generate flow-fields in the absence of individually trackable particles in the fluid. PIV has been used to measure flows of cytoplasm based on imaging subcellular vesicles and other features visible in the cytoplasm by transmitted light microscopy14.
<ol>
	<li>Download PIVLab, a MATLAB package for PIV, from the web site <a href="http://pivlab.blogspot.com/">http://pivlab.blogspot.com/</a>.</li>
	<li>Load the tiff image stack for each video sequence into the PIV program within MATLAB.</li>
	<li>Manually generate a mask for each image stack to mask out regions of the raw data outside the body of Stentor.</li>
	<li>Set PIVLab to use the fast-Fourier transform setting and a bin size of 64&#160;x 64&#160;pixels, which will bin the pixels in each frame into larger square pixels.</li>
	<li>Run PIVLab to compute cross-correlations between the pixels in each frame with nearby pixels in the next frame to yield a time-dependent flow vector centered at each binned pixel, which has the same time resolution as our raw data.</li>
	<li>Compute time averages of the resulting vector field output to generate streamlines for comparison between data taken with the Stentor before cutting and during wound healing, and calculate histograms of flow velocities (to study flow strength) and change of velocity at a given pixel over time (to study flow fluctuations).</li>
	<li>For image sequences with Stentor cleaved in half and cytoplasm visibly leaking into the surrounding buffer, calculate total flux of cytoplasm back into the cell body proper by multiplying the average flow velocity by the cross-sectional area of the base of the extracellular bubble of cytoplasm.</li>
</ol>

<ol>
	<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, 237-264 (2011).</li><li>Idone, V., Tam, C., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-way+traffic+on+the+road+to+plasma+membrane+repair.">Two-way traffic on the road to plasma membrane repair.</a> Trends Cell Biol. 18 (11), 552-559 (2008).</li><li>Reddy, A., Caler, E. V., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+repair+is+mediated+by+Ca2+-regulated+exocytosis+of+lysosomes.">Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes.</a> Cell. 106 (2), 157-169 (2001).</li><li>Foissner, I., Wastenays, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+characean+internodal+cell+as+a+model+system+for+studying+wound+healing.">The characean internodal cell as a model system for studying wound healing.</a> J. Microsc. 247 (1), 10-12 (2012).</li><li>Bement, W. M., Mandato, C. A., Kirsch, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound-induced+assembly+and+closure+of+an+actomyosin+purse+string+in+Xenopus+oocytes.">Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes.</a> Curr. Biol. 9 (11), 579-587 (1999).</li><li>Kim, H. G., Klotchkova, T. A., Kang, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+without+a+cell+membrane:+regeneration+of+protoplasts+from+disintegrated+cells+of+the+marine+green+alga+Bryopsis+plumosa.">Life without a cell membrane: regeneration of protoplasts from disintegrated cells of the marine green alga Bryopsis plumosa.</a> J. Cell Sci. 114 (11), 2009-2014 (2001).</li><li>Tartar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biology of Stentor. , 434 (1961).</li><li>Morgan, T. H. <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+proportionate+structures+in+Stentor.">Regeneration of proportionate structures in Stentor.</a> Biol. Bull. 2 (6), 311-328 (1901).</li><li>Heidemann, S. R., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+regional+approach+to+cell+mechanics.">Towards a regional approach to cell mechanics.</a> Trends Cell Biol. 14 (4), 160-166 (2004).</li><li>Kasza, K. E., Rowat, A. C., Liu, J., Angelini, T. E., Brangwynne, C. P., Koenderinck, G. H., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+as+a+material.">The cell as a material.</a> Curr. Opin. Cell Biol. 19 (1), 101-107 (2007).</li><li>Mitchison, T. J., Charras, G. T., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+a+poroelastic+cytoplasm+for+the+dynamics+of+animal+cell+shape.">Implications of a poroelastic cytoplasm for the dynamics of animal cell shape.</a> Sem. Cell Dev. Biol. 19 (3), 215-223 (2008).</li><li>De Terra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Stentor+coeruleus+on+Colpidium+campylum.">Culture of Stentor coeruleus on Colpidium campylum.</a> J. Eukar. Microbiol. 13 (3), 491-492 (1966).</li><li>Harris, E. 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+Chlamydomonas+Sourcebok.">The Chlamydomonas Sourcebok.</a> 1, (2009).</li><li>Vallotton, P., Ponti, A., Waterman-Storer, C. M., Salmon, E. D., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery,+visualization,+and+analysis+of+actin+and+tubulin+polymer+flow+in+live+cells:+a+fluorescent+speckle+microscopy+study.">Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study.</a> Biophys. J. 85 (2), 1289-1306 (2003).</li><li>Miyake, A., Harumoto, T., Iio, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defence+function+of+pigment+granules+in+Stentor+coeruleus.">Defence function of pigment granules in Stentor coeruleus.</a> Eur. J. Protistol. 37 (1), 7-88 (2001).</li></ol>

The authors declare that they have no competing financial interests.

The most important application of this method will be to use it to determine the molecular pathways that regulate cytoplasmic flow during wound healing in Stentor. A wide range of chemical inhibitors are available that target motor proteins as well as components of the cytoskeleton. Inhibitors of calcium signaling would also be likely candidates. It would also be of interest to remove or reduce the pigment granules (extrusomes) containing the blue pigment Stentorin, whose release from the cell is triggered under stress, and ask how the exocytotic release of Stentorin may affect flow of nearby cytoplasm. The pigment can be depleted using a simple bleaching treatment15 which could be done just prior to cutting. There does not appear to be any simple way to perform the wound healing assay in a parallel fashion since the cells need to be cut and imaged one at a time. Thus using a single microscope, it would be possible to operate on and measure cells at a rate of roughly 1 per 0.5 hr, so that during a single day of work it should be possible to obtain enough data points for a single compound to determine if it has a statistically significant effect on the process. While the method is thus not in any sense high throughput, it will be absolutely feasible to screen though a selected set of candidate compounds specific for a few carefully chosen target pathways.
A major limitation of the current approach is the relatively crude method for assembling the double-decker microscope. Assuming that the inverted microscope used for imaging is a general use instrument that other workers need to use for other purposes, an important consideration is the ease with which the apparatus can be disassembled and reassembled. While the present method using lab tape is indeed rapid and easily disassembled, it does require careful alignment by the user during assembly each time. An important modification would therefore be to construct a customized removable holder with slots for the eyepiece head of the inverted scope and the body of the dissecting scope. This should be easy to fabricate using modern 3D printer technology.
A second limitation of the approach is the reliance on DIC imaging of intracellular organelles as markers of flow. Because the identity of these organelles observed in the microscope is not currently known, it is not possible to know their size or potential interactions with other cellular components. It might therefore be advantageous to inject extrinsic tracers such as fluorescent beads and use them to track the flow. However this would require an extra step - microinjection - before the experiment could be done, thus further slowing the assay.
The final key limitation is the reliance on a single image plane for data acquisition. The primary importance of this assay is that it goes beyond simply asking whether a wound heals or not, and allows detailed analysis of the flow pattern to be obtained. In this regard, it will be extremely important to extend the imaging to three dimensional imaging. The current method images a single focal plane and thus when the PIV analysis is performed, the result is a slice through the full three dimensional flow field. This is informative but incomplete. By acquiring 3D images at each time step, it should be possible to obtain a fully three dimensional flow field. This will be particularly critical for observing exactly how the cytoplasm moves in the immediate vicinity of the wound site.

Regeneration and wound healing are important biological processes whose mechanisms remain areas of active investigation. Indeed, the ability to heal wounds and regenerate damaged parts is one of the features that sets living things apart from inanimate objects. Normally, regeneration is thought to be mediated by differentiation of stem cells into various cell types needed to rebuild a damaged or severed animal part, while wound healing is viewed in terms of new cells invading a wound to rebuild an intact epithelium. Thus both processes appear to be mediated by collectives of individual cells, and seem therefore to be quintessentially multicellular processes. It is therefore potentially surprising that individual cells are, in some cases, able to repair wounds and regenerate lost components1. Wound healing within single cells can involve multiple pathways including actin-myosin purse string contraction around the wound and membrane patching by rapid fusion of exocytotic vesicles1-3. Although many cell types are capable of some degree of wound healing4-6, perhaps the most dramatic example is the giant ciliate Stentor coeruelus7, shown in Figure 1. Individual Stentor cells are 1 mm long, and are covered with rows of cilia. Stentor cells are cone-shaped with an oral apparatus, consisting of thousands of cilia, at one end, and a narrow hold-fast structure at the other end. Stentor was a classical model organism for studying regeneration because if any part of the cell was cut off, it could regenerate7. For example, in a classic series of experiments, Thomas Hunt Morgan8 showed that a Stentor cell could be cut in half and each half would regenerate a normally proportioned cell in a matter of hours8. The molecular pathways responsible for regeneration in Stentor remain completely unknown.
The ability of Stentor cells to serve as a regeneration model system relies on the amazing ability of these cells to heal themselves after being cut. Why doesn&#39;t the cytoplasm of a Stentor cell leak out when the cell is cut? Wound healing is not due to immediate contraction of the cell cortex, because it is possible to take two Stentor cells, cut them, and then join the two cut cells together in various configurations7. During such grafting experiments, the cytoplasm of the two cells must remain exposed since it fuses when the cells are pushed together. Somehow, the cytoplasm is exposed enough to the surrounding media to allow grafting, but yet it does not flow out of the cell. Is the cytoplasm of Stentor sufficiently elastic that it simply cannot flow out even when a large hole is made in the plasma membrane? Do cells increase the viscosity of cytoplasm locally around a wound to prevent the cytoplasm from flowing out? Cytoplasm is a highly complex active material9-11, and the Stentor wound healing response represents a unique opportunity to study cytoplasmic flows and mechanics during single cell wound healing. However, the use of surgical methods to induce wounding in Stentor poses a technical challenge. In order to cut a 1 mm long cell using glass needles, the conventional surgical paradigm, it is necessary to operate at low magnification with long working distance. Under these imaging conditions, it is not possible to track the flow of cytoplasm, and all one can determine is whether the cytoplasm remains inside the cell or not. The real question we would like to ask is how the movement and viscosity of cytoplasm may change during cutting and healing. This requires high resolution imaging. Here we present an adaptation of Tartar&#39;s protocol for cutting Stentor7 that permits the cutting to be done under lower magnification optics to see the whole cell while simultaneously observing cytoplasm flow at high resolution, allowing this system to be used as a platform for exploring the mechanisms of single-cell wound healing.

<table><tbody><tr><td>&lt;em&gt;Stentor coeruleus&lt;/em&gt; live culture</td><td>Carolina Biological</td><td>131598</td><td></td></tr><tr><td>Methyl Cellulose, 1,500 cP</td><td>Sigma</td><td>M0387</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;EQUIPMENT:&lt;/p&gt;</td></tr><tr><td>Material Name</td><td>Company</td><td>Catalogue Number</td><td></td></tr><tr><td>Soft glass stirring rod 5 in long</td><td>Fisher Scientific</td><td>11-380A</td><td></td></tr><tr><td>Culture Dish 4.5 in 250 ml&amp;nbsp;glass</td><td>Carolina Biological</td><td>741004</td><td></td></tr><tr><td>IX81 inverted microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Axiovert 200M inverted microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Stemi 2000 Stereo Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Evolve 512 x 512 EMCCD camera</td><td>Photometrics</td><td></td><td></td></tr><tr><td>Plan Apo 20X objective</td><td>Olympus</td><td>UPLSAPO</td><td></td></tr></tbody></table>

visualizing cytoplasmic flow during single-cell wound healing in stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Using the protocols described above, it was found that the cytoplasm of Stentor is remarkably dynamic, both before and after cutting. Video 1 shows the extensive flow of cytoplasm present in un-cut cells. Despite our original expectation that Stentor&#39;s ability to survive cutting would involve a stiffening or gelling of cytoplasm, in fact when Stentor cells are cut with enough violence to draw the cytoplasm out of the cell, the cytoplasm remains highly dynamic and manages to flow back into the cell in a highly directed fashion (Video 2).
In addition to demonstrating a previously unsuspected degree of cytoplasmic motion during wound healing, the high resolution of imaging in the assay can be used to quantify the flow rates using particle image velocimetry. The ability to make such measurements will allow the effect of chemical inhibitors to be rigorously tested.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50848/50848fig1highres.jpg" src="/files/ftp_upload/50848/50848fig1.jpg" /> 
Figure 1. Morphology of a living Stentor cell. Darkfield image of a living Stentor cell that has been fed with Chlamydomonas as described in Procedure 1.3, visible as green material inside food vacuoles. The oral apparatus through which the cell eats is visible as a rim of cilia at the top of the image.
<a href="https://www.jove.com/files/ftp_upload/50848/50848fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50848/50848fig2highres.jpg" src="/files/ftp_upload/50848/50848fig2.jpg" /> 
Figure 2. Preparation of glass needle for surgery. (A) Needle is drawn from the end of a solid glass rod. (B) High magnification view of needle tip indicating the length and sharpness necessary for surgery.
<a href="https://www.jove.com/files/ftp_upload/50848/50848fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50848/50848fig3highres.jpg" src="/files/ftp_upload/50848/50848fig3.jpg" /> 
Figure 3. Custom coverslip holder for microsurgery. In order to allow open access for surgery while allowing optimal imaging conditions, cells are mounted on a coverslip rather than a thick class slide. In order to allow the coverslip to fit on the microscope stage a plastic holder can be fabricated using a 3D printer. The holder has the same dimensions as a standard microscope slide while the depression in the middle matches the dimensions of the coverslip. A hole in one corner allows the coverslip to be popped out of the holder.
<a href="https://www.jove.com/files/ftp_upload/50848/50848fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50848/50848fig4highres.jpg" src="/files/ftp_upload/50848/50848fig4.jpg" /> 
Figure 4. Double-decker microscope configuration. A dissecting microscope head is mounted on top of the eyepiece head of an inverted microscope. Lab tape allows the apparatus to be rapidly assembled and disassembled when the experiment is completed.
<a href="https://www.jove.com/files/ftp_upload/50848/50848fig4highres.jpg" target="_blank">Click here to view larger image</a>.
Video 1. Example of cytoplasmic flow observed in living Stentor cell. Cell imaged with 10X objective with darkfield optics at a rate of 2 frames/sec.
Video 2. Imaging of a Stentor cell as it is cut and disrupted with a glass needle. After cutting a substantial quantity of cytoplasm has been pulled out of the main cell body, but it rapidly flows back into the cell.

Watch this Scientific Journal Video about Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus at JoVE.com

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

visualizing-cytoplasmic-flow-during-single-cell-wound-healing-stentor

Research

JoVE Journal

Biology

5.7K Views.  Marine Biological Laboratory. The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells. By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Video: Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Developmental Biology

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Medicine

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Immunology and Infection

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

Biochemistry

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes&#8212;many not associated with the oral apparatus (OA)&#8212;that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video