This work was supported, in part, by Marine Biological Laboratory Whitman Early Career Fellowship (JYS). We acknowledge Evan Burns, Mit Patel, Melanie Melo, and Skylar Widman for helping with some of the preliminary analysis and code testing. We thank Mark Slabodnick for discussion and suggestions. WFM acknowledges support from NIH grant R35 GM130327.

NOTE: A population of approximately one hundred S. coeruleus cells were cultured in accordance with a previously published JoVE protocol8.
1. Sample preparation
<ol>
	<li>Cut a piece of 250 &#181;m-thick silicone spacer sheet (Table of Materials) slightly smaller in both height and width than a microscope slide. Using a 5/16&#34; hole punch, create circular wells. Be mindful of leaving sufficient space between neighboring wells to ensure a good seal. 
	NOTE: A space of 3 mm between neighboring wells was found to be sufficient. With practice, ten wells can be placed on a single sample slide.</li>
	<li>Initiate regeneration of OA by incubating the cells in 10% sucrose or 2% urea for 2 min (Figure 1A). Then, wash three times with fresh medium8. Gently pipette approximately ten Stentor into each well. Be careful of matching sample volume to the well volume as closely as possible. 
	NOTE: For the well dimensions used here, 12.5 &#181;L of sample was pipetted into each well.</li>
	<li>Close the wells by gently lowering a piece of coverglass (see Table of Materials) over the wells starting from one edge. Use a narrow and slightly flexible object, such as a 10 &#181;L pipette tip, to press down on the coverglass where it contacts the silicone sheet, to ensure a good seal.</li>
</ol>
2. Visible light microscopy time-lapse
<ol>
	<li>Place the sample on the microscope stage, and set magnification to the lowest available such that one well fits in frame in its entirety. 
	NOTE: A 1.6x camera adaptor and 1x magnification on the objective were used on the microscope for this protocol.</li>
	<li>Begin time-lapse. Acquire a 10 s video at 30 frames per second of each well at each timepoint. If using a microscope setup with a motorized X-Y stage, automate the entire time-lapse. Otherwise, ensure that a user is present at each timepoint to manually translate the sample to record each well. 
	NOTE: Avoid leaving the sample illuminated when not imaging to avoid heating and evaporation. The sample volumes are small, and evaporation will lead to air bubbles.</li>
	<li>Save movies as TIFF, MOV, or AVI. 
	NOTE: These are the most common non-proprietary video file types. Depending on the specific microscope software, the videos may save by default to a proprietary file type, but then can be exported to one of the aforementioned file types.</li>
	<li>Use a pixel to millimeter conversion factor for physical scale, and perform a calibration or use known pixel size from the camera used. To calibrate, acquire a clear image of a calibration slide or a clear ruler at the same magnification settings as the videos. Using any image viewing program, count the number of pixels between marks of a known physical distance. 
	NOTE: For example, if the image of a ruler shows the 1 mm mark and 2 mm mark to be separated by 100 pixels, the conversion factor is 1 mm per 100 pixels. Alternatively, to derive this factor from camera pixel size, simply multiply the camera pixel size by the magnification. For example, if the camera used has 3.45 &#181;m pixels, and the magnification used was 1.6x, then the conversion is 3.45 &#181;m * 1.6 = 5.52 &#181;m per 1 pixel.</li>
</ol>
3. Cell tracking
<ol>
	<li>Download the two scripts, TrackCells.m and CleanTraces.m, to an easy-to-remember location on the computer. If the data videos are not already on this computer, transfer them onto this computer. 
	NOTE: The data videos and the scripts do not need to be in the same folder.</li>
	<li>Organize data videos into folders, one for each timepoint. Use the script TrackCells.m first to perform automated cell tracking in the data videos. Open TrackCells.m and run the script.</li>
	<li>Choose Add to Path if prompted by a pop-up window., which will typically only happen when the script is run for the first time from a given folder. When prompted, Select One or More Data Videos to Initiate Tracking, navigate to the data videos (section 2). Select multiple video files by using shift-click, control-click, or by holding down the left mouse button while moving over the files to highlight them.</li>
	<li>Once satisfied with the list of files in the File name: box at the bottom of the prompt window, click on Open. Perform a test run on one video first to confirm all parameters are set correctly (see discussion below). 
	&#8203;NOTE: This script will now create a folder for each video file chosen. It will then write into this folder each frame of the video as a .jpg file and a file named position_estimates.mat, which contains all traces found in the video. Depending on the size of the videos, the number of videos, and the speed of the computer, this script can take hours to run.</li>
</ol>
4. Trace verification
<ol>
	<li>Verify that steps described in section 3 were followed correctly by checking that there are no error messages before proceeding. Use the CleanTraces.m script to manually reject anomalous or false traces. Open CleanTraces.m in the MATLAB editor window by double-clicking on the file.</li>
	<li>At the prompt Select data folder output by TrackCells.m. It will have the same name as the video file, navigate to one of the folders created as described in section 3. Choose only one folder. 
	NOTE: This script will now display the traces one-by-one in a pop-up window. They are overlaid on the frame of the video where the trace starts, in green, and the frame of the video where the trace ends, in magenta. Therefore, a valid trace should link a green cell and a magenta cell.</li>
	<li>When prompted, enter 1 to keep the trace and 0 to reject the trace. Press Enter to move on to the next trace. 
	NOTE: New traces will continue to display until either there are no more traces, or the first sixty have been shown. When this process is complete, the script will automatically create a folder named CLEAN TRACES for saving the outputs and display all remaining traces on top of the first frame of the video (Figure 1B). This image is automatically saved as LabeledTraces.png for future reference. All traces the user had chosen to keep will be saved in the file clean_traces.mat.</li>
	<li>Complete this step for all videos in one time point before continuing. 
	&#8203;NOTE: For the data in this manuscript, one video was acquired for each well at each timepoint, for a total of ten videos per timepoint.</li>
</ol>
5. Data visualization
NOTE: To visually compare the motility of the entire cell population across different timepoints, all traces from section 4 were translated to begin at the origin and create one radial displacement versus time plot for each timepoint (Figure 1C-F, see Supplemental Figure S1 for all time points).
<ol>
	<li>Begin by downloading the script titled SpaghettiPlots.m. Open and run the script. When a window file browser window pops up with the prompt Select time folder containing well data (clean_traces.mat) to graph, navigate to the folder of one of the time points. Note that this folder should contain within it a folder for each of the analyzed videos.</li>
	<li>When prompted Calibration: What is the number of pixels per millimeter?, type in the calibration value found in step 2.4, and press Enter. 
	NOTE: The script will now combine the traces from all the analyzed videos at this time point into a single plot (Figure 1C-F). Faint dotted circles in the plot indicate radial displacements of 1, 2, 3, and 4 mm.</li>
	<li>Adjust axes of the plot as necessary by changing line 55 of the script, which by default, is set to rr = 4 for including a radius of up to 4 mm. Save the plot.</li>
</ol>

<ol>
	<li>Lillie, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+smallest+parts+of+stentor+capable+of+regeneration;+a+contribution+on+the+limits+of+divisibility+of+living+matter.">On the smallest parts of stentor capable of regeneration; a contribution on the limits of divisibility of living matter.</a> Journal of Morphology. 12 (1), 239-249 (1896).</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> The Biological Bulletin. 2 (6), 311-328 (1901).</li><li>Tartar, V., Kerkut, G. A. <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. , (1961).</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=Reactions+of+Stentor+coeruleus+to+certain+substances+added+to+the+medium.">Reactions of Stentor coeruleus to certain substances added to the medium.</a> Experimental Cell Research. 13 (2), 317-332 (1957).</li><li>Kelleher, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+kinetic+model+for+microtubule+polymerization+during+oral+regeneration+in+Stentor+coeruleus.">A kinetic model for microtubule polymerization during oral regeneration in Stentor coeruleus.</a> Biosystems. 9 (4), 269-279 (1977).</li><li>Slabodnick, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinase+regulator+Mob1+acts+as+a+patterning+protein+for+Stentor+morphogenesis.">The kinase regulator Mob1 acts as a patterning protein for Stentor morphogenesis.</a> PLOS Biology. 12 (5), 1001861 (2014).</li><li>Wan, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reorganization+of+complex+ciliary+flows+around+regenerating+Stentor+coeruleus.">Reorganization of complex ciliary flows around regenerating Stentor coeruleus.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 375 (1792), 20190167 (2020).</li><li>Lin, A., Makushok, T., Diaz, U., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+study+of+regeneration+in+Stentor.">Methods for the study of regeneration in Stentor.</a> Journal of Visualized Experiments JoVE. (136), e57759 (2018).</li><li>Sood, P., McGillivary, R., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+program+of+regeneration+in+the+giant+single+cell,+Stentor+coeruleus.">The transcriptional program of regeneration in the giant single cell, Stentor coeruleus.</a> bioRxiv. , 240788 (2017).</li><li>Onsbring, H., Jamy, M., Ettema, T. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+sequencing+of+Stentor+cell+fragments+reveals+transcriptional+changes+during+cellular+regeneration.">RNA sequencing of Stentor cell fragments reveals transcriptional changes during cellular regeneration.</a> Current Biology. 28 (8), 1281-1288 (2018).</li></ol>

The authors have nothing to disclose.

Many particle and cell tracking algorithms currently exist, some entirely free. Cost and user-friendliness are often trade-offs requiring compromise. Additionally, many of the existing cell-tracking programs are designed to track slow crawling motion of tissue culture cells, rather than the fast swimming motion of Stentor, which rotates while swimming and can undergo sudden changes of direction. After testing many of these options, the protocol presented here is intended to be a one-stop solution to go all the w...

Stentor coeruleus (Stentor) is a well-known model organism that has been used to study unicellular regeneration owing to its large size, ability to withstand several microsurgical techniques, and ease of culturing in a laboratory setting1,2,3. Early regeneration studies focused on the largest and most morphologically distinct feature in Stentor&#8212;the OA&#8212;which is shed completely upon chemical shock4,5,6. De novo replacement of a lost OA begins with the emergence of a new membranellar band&#8212;an array of cilia that gradually shift towards the anterior of the cell before forming a functional OA over eight morphological stages3. These stages have been observed sequentially, regardless of temperature, and provide a universal reference point for nearly all studies5.
Mechanistic analysis of Stentor regeneration requires tools for measuring the timing of regeneration that are robust and simple enough to be applied to multiple samples as part of a chemical or molecular screen. The standard method for performing a cell-based assay is imaging, in this case, imaging the formation of new OA during regeneration. However, such imaging-based assays are most effective when the regenerating structure contains distinct molecular components that can be used as markers, so that they would be easily detected in a fluorescence image. In the case of the Stentor OA, the known components (cilia, basal bodies) are also present on the rest of the cell surface; therefore, recognizing the restoration of the OA cannot be achieved simply by looking for the presence or absence of a component.
Rather, some form of shape recognition would be required to detect an OA, and this is potentially very challenging given the fact that Stentor cells often change shape via a rapid contractile process. This paper presents an alternative assay for regeneration that relies on the motile activity of the body and OA cilia. As the OA regenerates, the newly formed cilia undergo reproducible changes in position and activity, which in turn, affects the swimming motility of the cell. By analyzing motility, it is possible to perform an assay for &#34;functional regeneration&#34; that quantifies regeneration by quantifying the function of the regenerated structures. Previous analysis of Stentor ciliary function during regeneration used particle image velocimetry, combined with tracer beads added to the external media, to observe changes in flow pattern at different stages of regeneration7; however, this approach requires laborious imaging of individual cells and their associated flow fields, one at a time.
By using the motion of the cell itself as a proxy for cilia-generated flow, it would be possible to analyze larger numbers of cells in parallel, using low-resolution imaging systems compatible with high-throughput screening platforms. This assay can, in principle, be used to study development and functional regeneration in other swimming organisms in the hundreds of microns to millimeters size scale. Section 1 of the protocol describes the construction of a multiwell sample slide, which allows for high-throughput imaging of a population of cells over up to an entire day. Details are provided for how to adjust for use with other cell types. Section 2 of the protocol covers the acquisition of video data for this assay, which can be accomplished on a dissection microscope with a digital single-lens reflex camera. Section 3 of the protocol provides a walk-through of cell tracking and cell speed calculation using MATLAB code (Supplemental Information). Section 4 of the protocol explains how to turn the numerical results into plots as shown in Figure 1C-F and Figure 2C for easy interpretation of results.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25 mm-thick silicone sheet</td>
			<td>Grace Bio-Labs</td>
			<td>CWS-S-0.25</td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 50 mm, #1.5 coverglass</td>
			<td>Fisher Scientific</td>
			<td>NC1034527</td>
			<td>As noted in Discussion, smaller coverglass can be used if fewer sample wells are placed on one slide.</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td></td>
			<td></td>
			<td>We used Nikon D750</td>
		</tr>
		<tr>
			<td>Chlamydomonas 137c WT strain</td>
			<td>Chlamydomonas Resource Center</td>
			<td>CC-125</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MATHWORKS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB Image Processing Toolbox</td>
			<td>MATHWORKS</td>
			<td></td>
			<td>needed for TrackCells.m and CleanTraces.m</td>
		</tr>
		<tr>
			<td>MATLAB Statistics and Machine Learning Toolbox</td>
			<td>MATHWORKS</td>
			<td></td>
			<td>needed for TrackCells.m</td>
		</tr>
		<tr>
			<td>Microscope with camera port</td>
			<td></td>
			<td></td>
			<td>We used Zeiss AxioZoom v1.6 and Leica S9E</td>
		</tr>
		<tr>
			<td>Pasteurized Spring Water</td>
			<td>Carolina</td>
			<td>132458</td>
			<td></td>
		</tr>
		<tr>
			<td>TAP Growth Media</td>
			<td>ThermoFisher Scientific</td>
			<td>A1379801</td>
			<td>Can also be made for much cheaper following recipe from Chlamy Resource Center</td>
		</tr>
	</tbody>
</table>

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.

The goal of this assay is to quantify the gradual change of movement patterns and phased increase in movement speed from cells within a large (N ~100) regenerating Stentor population. To aid interpretation of results, the custom code included in this protocol generates two types of plots: an overlay of all cell movement traces in a set of video data (Figure 1C-F and Figure S1), and a plot of swim speed by hour since the start of reg...

Watch this Scientific Journal Video about Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking at JoVE.com

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.

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 ...

analysis-motility-patterns-stentor-during-after-oral-apparatus

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JoVE Journal

Biology

1.7K Views.  Scripps, Pitzer, and Claremont McKenna of The Claremont Colleges. 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.

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

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

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.

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.

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

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...