We would like to thank Dr. Guo-Chiuan Hung and Dr. Bharatkumar Joshi for valuable comments that improved the manuscript.

All the experiments conducted in these studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Food and Drug Administration (FDA) research facility.
1. Isolation and Culture of Mouse Embryonic Fibroblasts (MEFs)
<ol>
	<li>Isolate mouse embryonic fibroblasts (MEFs) from a PP2A-B56&#947;- mouse strain and wild type littermates by standard protocol 7,8,9.</li>
	<li>ExpandMEFs for 3 passages, freeze and store until needed for experiments 8.</li>
</ol>
2. Culturing Mefs in 2-Well Chambered Cover Glass for Live Imaging
<ol>
	<li>Prepare 500 mL of MEF growth media containing Dulbecco Modified Eagle Medium (DMEM/F12) with 10% Fetal Bovine Serum (FBS), 1 X Penicillin/Streptomycin antibiotics, L-Glutamine (200 mM) and 1X non-essential amino acids (NEAA) in a 500-mL media bottle.</li>
	<li>Thaw vials of frozen MEFs from wild type and PP2A-B56&#947;- mice at passage 3 in pre-warmed water bath at 37 &#176;C.</li>
	<li>Transfer thawed MEFs to 15-mL tubes and slowly add dropwise 20 mL of DMEM/F12 media to the 15-mL tubes using a 10-mL pipette.</li>
	<li>Transfer MEFs along with 20 mL of DMEM/F12 growth media in T75 flasks and expand them until the cells are 70% confluent. Confluency is estimated using an inverse microscope at 4x or 10x magnification.</li>
	<li>Aspirate the growth medium using a glass pasture pipette attached to a vacuum system in the hood, add 3 mL of 0.25% trypsin/EDTA and incubate at 37 &#176;C for 5 min. Add 3 mL of DMEM/F12 growth medium to stop the reaction.</li>
	<li>Centrifuge the cells at low-speed (300 x g) for 5 min at room temperature. Carefully remove the supernatant and re-suspend the cell pellet in 1 mL of DMEM/F12 growth medium pre-warmed at 37 &#176;C in a water bath.</li>
	<li>Enumerate cells using the trypan blue exclusion method or other appropriate cell counting method 8.</li>
	<li>Seed approximately 20,000 MEFs/well in a 2-well chambered cover glass. Add 200 &#181;L/well DMEM/F12 growth medium and allow the cells to attach by incubating them overnight at 37 &#176;C and 5% CO2.</li>
</ol>
3. Synchronization
<ol>
	<li>Synchronize MEFs in G0/G1 phase by incubating cells in DMEM/F12 growth media containing 0.1% FBS for 24 h, to obtain a maximum number of cells in G0/G1 phase. 
	Note: Although serum starvation was used as a method of preference, various other methods can be used depending on the mitotic stage at which cells are needed to be arrested 10.</li>
</ol>
4. Labeling
<ol>
	<li>Prepare 1.5 mg/mL stock solution of nocodazole in DMSO. Nocodazole arrests cells at G2/M phase by inhibiting microtubule formation 10.</li>
	<li>Three days post synchronization, add 200 &#181;L of growth medium (with 10% FCS), 200 ng/mL nocodazole and CL-HB regent (Histone 2B-GFP BacMam 2.0).</li>
	<li>Calculate the CL-HB regent Particles Per Cell (PPC) to be added to cells as follows: 
	<img alt="Equation 1" src="/files/ftp_upload/57389/57389eq1.jpg" /> 
	Where the number of cells is the estimated total number of cells at the time of labeling, PPC is the number of Particles Per Cell, and 1 &#215; 108 is the number of particles per mL of the reagent. For example, to label 20,000 cells with a PPC of 30 
	<img alt="Equation 2" src="/files/ftp_upload/57389/57389eq2.jpg" /> 
	NOTE: CL-HB regent works well with most cell types between 10 and 50 PPC. However, 30 PPC worked best for this study.</li>
	<li>Incubate MEFs for 18 h at 37 &#176;C and 5% CO2. 
	NOTE: For the current experiment, visualization of chromosomes in mitotic cells escaping the spindle assembly checkpoint occurred 18 h after cell cycle arrest with nocodazole.</li>
</ol>
5. Visualization of Lagging Chromosomes
<ol>
	<li>Visualize mouse embryonic fibroblasts (MEFs) using a spinning disk confocal microscope system equipped with an environmental chamber and an oil immersion, 63x objective lens.
	<ol>
		<li>Use excitation wavelength of 488 nm and emission wavelength 450 nm for GFP channel image acquisition.</li>
		<li>Use a spinning disk confocal microscope system with the capability of motorized scanning stages, Z-Piezo inserts, stage-top incubation and direct fluorescence recovery after photo bleaching for this technique.</li>
	</ol>
	</li>
	<li>One day before imaging, turn the environmental chamber power ON and warm up the entire chamber at 37 &#176;C overnight.</li>
	<li>Turn power ON for the microscope stand, camera, spinning disk unit, illuminator, argon laser, computer and motorized stage.</li>
	<li>Let the system warm up for 3 min; start the argon laser by turning on ignition key. Switch the toggle switch for argon laser from &#34;standby&#34; to &#34;laser run&#34;.</li>
	<li>Launch the data acquisition and processing software.</li>
	<li>Initiate the CO2 controller for the stage top incubator and set the concentration of CO2 at 5%. This must be done before commencement of imaging.</li>
	<li>Remove the chambered cover glass from the incubator and place on the stage for visualization. Visualize cells via an oil immersion 63x objective lens (NA1.4).</li>
	<li>View through the ocular lenses, focus the image and identify a cell in nuclear envelope breakdown (NEBD) stage.</li>
	<li>Initiate appropriate laser (488 nm argon laser to visualize Histone2B-GFP).</li>
	<li>Open acquisition control window and set exposure time for the GFP channel. Identify a cell that is in NEBD.
	<ol>
		<li>Manually determine the target cell&#39;s top and bottom focal plane, and enter the xyz optical sectioning settings.</li>
		<li>Observe it for 20 min; if the cell does not proceed through cell division, stop image acquisition after 20 min and move on to the next cell that is in NEBD.</li>
	</ol>
	</li>
	<li>Approximately 1 h per cell is needed to image lagging chromosomes during mitosis in PP2A-B56&#947;- cells that escaped from the SAC. To obtain data for a movie, take pictures every 3 min. 
	NOTE: Wild type cells arrest and do not progress past NEBD when treated with nocodazole.</li>
	<li>Save images in zvi file format for further analysis.</li>
</ol>
6. Image Processing and Analysis
Note: Perform three-dimensional image processing and analysis using any available software such as Axiovision version 4.8.2 or Imaris version 8.2. For this study the ImageJ software was used.
<ol>
	<li>Open the image sequence. If the images are already in a stack format, proceed to the next step. If not, combine all relevant images into a stack using &#39;Image &#62; Stacks &#62; Images to Stack&#39; in the menu bar.</li>
	<li>Perform any adjustments as required to brightness/contrast and levels.</li>
	<li>To add a time stamp to the movie:
	<ol>
		<li>Go to &#39;Image &#62; Stacks &#62; Label&#8230;&#39; in the menu bar.</li>
		<li>Select the appropriate format, the starting time value, and time interval between each image.</li>
		<li>Check the &#39;Preview&#39; box and adjust location and format settings. Press &#39;OK&#39; to apply the time stamp.</li>
	</ol>
	</li>
	<li>To add a scale bar to the movie:
	<ol>
		<li>Set the image scaling under &#39;Analyze &#62; Set Scale&#39; in the menu bar.</li>
		<li>In the &#39;Distance in pixels&#39; field, enter a number of pixels where the distance is known, and in the &#39;Known distance&#39; field enter the distance. Set the correct unit of length for the distance, e.g., in &#181;m. Press &#39;OK&#39; to apply the scale to the stack.</li>
		<li>Go to &#39;Analyze &#62; Tools &#62; Scale Bar&#8230;&#39; to add the scale bar. Set the size of the bar as &#39;Width in &#181;m&#39;, and adjust the remaining formatting options as appropriate. Check the &#39;Label all slices&#39; box to add the scale bar to the entire stack.</li>
	</ol>
	</li>
	<li>Preview the movie by clicking on the triangular play button on the bottom left edge of the image window. Adjust the frame rate using &#39;Image &#62; Stacks &#62; Animation &#62; Animation Options&#39; in the menu bar.</li>
	<li>Export the movie file by selecting &#39;File &#62; Save As &#62; AVI&#8230;&#39; and select the frame rate and compression.</li>
	<li>Press &#39;OK&#39; to choose the save location and file name. Press &#39;Save&#39; to save the movie file.</li>
</ol>

<ol>
	<li>Funk, L. C., Zasadil, L. M., Weaver, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+in+CIN:+Mitotic+Infidelity+and+Its+Consequences+for+Tumor+Promotion+and+Suppression.">Living in CIN: Mitotic Infidelity and Its Consequences for Tumor Promotion and Suppression.</a> Dev Cell. 39, 638-652 (2016).</li><li>Etemad, B., Kops, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+issues:+kinetochore+transformations+and+spindle+checkpoint+silencing.">Attachment issues: kinetochore transformations and spindle checkpoint silencing.</a> Curr Opin Cell Biol. 39, 101-108 (2016).</li><li>Lara-Gonzalez, P., Westhorpe, F. G., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spindle+assembly+checkpoint.">The spindle assembly checkpoint.</a> Curr Biol. 22, 966-980 (2012).</li><li>Jallepalli, P. V., Lengauer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+segregation+and+cancer:+cutting+through+the+mystery.">Chromosome segregation and cancer: cutting through the mystery.</a> Nat Rev Cancer. 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The authors declare that they have no competing financial interests.

Cell cycle control checkpoints that ensure accurate chromosome segregation prevent aneuploidy and cell transformation 1,2,3. In the present study, we found that inactivation of PP2A-B56&#947; resulted in a weakened spindle assembly checkpoint. Live cell imaging allowed us to observe chromosomal mis-segregation during mitosis in PP2A-B56&#947; MEFs treated with nocodazole 9.
This protocol utilizes Histone2B-GFP label generated by BacMam 2.0 technology to label chromosomes for live cell imaging. One of the critical steps in labeling is calculating right amount of particles per cells (PPC) to achieve enough labeling. It was found through titration that 10-50 PPC works best. This reagent could be toxic to the cells; hence it is necessary to titrate PPC used to achieve balance between labelling and toxicity to the labelled cells. Also, this is a transient transfection and can be detected only up to 5 days, hence, it may not be appropriate for studies that would warrant longer observation periods.
Traditional tagging of the protein or DNA can require complicated protocols, but here we use a reagent which requires only an overnight incubation with desired cells. In addition, live cell imaging was performed using spinning disc confocal microscopy which unlike confocal laser scanning microscopy is faster and reduces phototoxicity and bleaching effects in the living cells 11. This protocol allows easy, convenient method to label mitotic cells and visualize chromosomal segregation as compared to the other techniques such as FISH, immunohistochemistry and flow cytometry. This protocol also allows the capture of lagging chromosomes images throughout mitosis providing additional information to that obtained using traditional immunocytochemistry, FISH or flow cytometry, thereby making it possible to capture the phenotypes such as lagging chromosome in both space and time. This protocol allows convenient, fast and easy labeling of chromosomes in wide variety of cell lines, primary cells, stem cells, neurons and immortalized T-cells 12,13,14.
Various types of cell therapy products are being developed with the hopes to treat varied disease conditions 15. One of the challenges faced in the development of these cell therapies involves safety concerns due to possibility of genetic instability and tumorigenesis. This protocol describes a convenient and easy method to label chromosomes in variety of cell types. In the future it could be employed to develop an assay to study and measure genetic instability and quality of the cell therapy products.

In the following protocol, we describe a convenient method to visualize the chromosomal segregation and mitotic progression during cell cycle in mouse embryonic fibroblasts using Histone 2B-GFP, BacMam 2.0 labeling and live cell imaging.
Cell cycle control checkpoints monitor chromosome segregation and play an important role in the maintenance of the genetic integrity of the cell 1,2,3. Accumulation of mis-segregated chromosomes can lead to aneuploidy, which is a hallmark of most solid tumors 4. Hence, detection of lagging chromosomes can be used as a method to study chromosomal instability.
Fluorescently labeled proteins can be used to visualize live chromosome segregation and chromosomal lagging but the generation of mCherry-tagged or H2B-GFP tagged protein requires substantial knowledge of gene delivery and molecular biology 5. Here we describe the use of CellLight Histone2B-GFP BacMam 2.0 reagent, hereafter called CL-HB regent, for the sake of simplicity. This reagent can be used immediately and thus eliminates concerns about vector quality and integrity. In addition, this reagent does not require the use of potentially harmful treatments or lipids and dye-loading chemicals. Unlike conventional fluorescent labels, the CL-HB regent stains independently of function (i.e., membrane potential). The CL-HB regent can be simply added to the cells and incubated overnight for protein expression. The CL-HB regent does not replicate in mammalian cells and can be used in biosafety level (BSL) 1 laboratory settings. Also, this transient transfection can be detected after overnight incubation for up to 5 days, enough time to carry out most dynamic cellular analyses.
Alternatively, chromosomal abnormalities could be studied by various techniques such as flow cytometry, immunohistochemistry or fluorescence in situ hybridization (FISH) 6. Flow cytometry can be used to study aneuploidy, which can be measured based on DNA content and the phase of cells in the cell cycle. Although flow cytometry can be used to measure aneuploidy, it does not provide information on chromosomal mis-segregation. FISH and immunohistochemistry techniques use fluorescent probes to bind to DNA or chromosomes. While these techniques provide a snapshot of the status of a population of cells, they do not allow live cell imaging thereby missing any information obtained through spatiotemporal visualization of cytokinesis in specific cells followed over a period of time.
This protocol was used to study lagging chromosomes or chromosomal mis-segregation in nocodazole treated mouse embryonic fibroblasts (MEFs) isolated from PP2A-B56&#947;- mice. In addition to above application, this protocol provides a simple tool to label and visualize chromosomal segregation in various cell types which can be used to study cell cycle regulation or chromosomal instability in tumor cells. In addition, it can also be used to study chromosomal instability caused by various drug treatments or to study the effects of gene knock out resulting in chromosomal mis-segregation.

<table border="0" cellpadding="0" cellspacing="0" style="width:644px;" width="645">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">DMEM/F12</td>
			<td style="width:135px;">Gibco</td>
			<td align="right" style="width:128px;">11320082</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Gibco</td>
			<td align="right">25030081</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">MEM Non-Essential amino acids solution (100X)</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">11140050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Dulbecco&#8217;s Phosphate Buffered saline</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Trypsin-EDTA</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Fetal bovine serum</td>
			<td style="width:135px;">Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td style="width:135px;">EMD Millipore</td>
			<td>MX 1458-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryogenic vial storage boxes</td>
			<td style="width:135px;">Fisherbrand</td>
			<td style="width:128px;">10-500-28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Cryogenic vials</td>
			<td style="width:135px;">Corning/costar</td>
			<td align="right">431416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">T75 flasks</td>
			<td style="width:135px;">Cellstar</td>
			<td align="right" style="width:128px;">658175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">2 well chambered cover glass</td>
			<td style="width:135px;">Nunc</td>
			<td style="width:128px;">155380PK</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Cellometer Vision Cell Profiler</td>
			<td style="width:135px;">Nexcelom Bioscience LLC</td>
			<td style="width:128px;">Cellometer Vision Trio</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Nocodazole</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:128px;">M1404</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">CellLight Histone 2B-GFP, BacMam 2.0</td>
			<td style="width:135px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:128px;">C10594</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:191px;">Zeiss Cell Observer Spinning Disk Confocal Microscope system</td>
			<td style="width:135px;">Carl Zeiss Microscopy</td>
			<td style="width:128px;">Zeiss Cell Observer SD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Water Bath</td>
			<td style="width:135px;">Thermoscientific</td>
			<td style="width:128px;">280 series</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Incubator</td>
			<td style="width:135px;">Sanyo commercial solutions</td>
			<td style="width:128px;">MCO-18AIC (UV)</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Class II Biological safety cabinet</td>
			<td style="width:135px;">The Baker Company</td>
			<td style="width:128px;">SterilGard</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Axiovision software</td>
			<td style="width:135px;">Zeiss</td>
			<td style="width:128px;">Ver.4.8.2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">ImageJ software</td>
			<td style="width:135px;">National Institute of Health</td>
			<td style="width:128px;">Ver. 1.51r</td>
			<td></td>
		</tr>
	</tbody>
</table>

live cell imaging of chromosome segregation during mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

MEFs from wild type and PP2A-B56&#947;- mice were seeded in a 2-well chamber cover glass and allowed to attach. On day 2, MEFs were synchronized using 0.1% FBS for 24 h. On day 3, MEFs in media with 200 ng/mL nocodazole and 30 PPC CL-HB regentwere incubated for 18 h at 37&#176;C and 5% CO2. On day 4, the cells were imaged using a spinning disk confocal microscope system (Figure 1). Live cell imaging was used to visualize the fate of individual cells as they progressed from NEBD through mitosis (Figure 2). Nocodazole is known to arrest the wild type cells at G2M phase by interfering with microtubule formation. It was observed that post nocodazole treatment wild type cells were arrested at metaphase upper panel (Figure 2). We observed that lack of PP2A-B56&#947; weakened the cell cycle control, which allowed PP2A-B56&#947;- MEFs to overcome cell cycle arrest induced by nocodazole 9. In addition, abnormal chromosomal segregation was detected in 62% (13 out of 21) of mitotic PP2A-B56&#947;- MEFs treated with nocodazole (shown by yellow arrows in lower panel in Figure 2). Five of the PP2A-B56&#947;- MEFs arrested at NEBD and 3 went through mitosis without observable defects. In contrast, all 21 wild type cells that were imaged did not proceed through mitosis and were found to be arrested by metaphase (upper panel Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57389/57389fig1.jpg" /> 
Figure 1. Basic flowchart for live cell imaging. MEFs from wild type and PP2A-B56&#947;- mice were seeded in a 2-well chambered cover glass overnight. Day 2, cells were synchronized using 0.1% FBS for 24 h. Day 3, cells were treated with 200 ng/mL nocodazole in growth media and 30 PPC Histone 2B-GFP, BacMam 2.0 reagent for 18 h. Day 4 the cells were imaged using spinning disk confocal microscope system. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57389/57389fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57389/57389fig2.jpg" /> 
Figure 2. Chromosomal abnormalities are detected in PP2A-B56&#947;- MEFs. Time lapse microscopy live images captured at various time points in mitosis (0-51 min) of nocodazole treated wild type and PP2A-B56&#947;- MEFs. The wild type cells were found to be arrested at metaphase (upper panel), whereas PP2A-B56&#947;- MEFs were able to go through mitosis (lower panel). Chromosomal lagging or mis-segregation can be seen in PP2A-B56&#947;- MEFs shown by yellow arrows in the lower panel. Scale bar = 5 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57389/57389fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Live Cell Imaging of Chromosome Segregation During Mitosis at JoVE.com

Live Cell Imaging of Chromosome Segregation During Mitosis

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

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Cell Division

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9.2K Views.  US Food and Drug Administration. This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Video: Live Cell Imaging of Chromosome Segregation During Mitosis

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...