Name: live cell imaging of chromosome segregation during mitosis
Uploaded: 2018-03-14T15:00:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of Chromosome Segregation During Mitosis at JoVE.com

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,<sup class=...

<ol>
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S., Narita, T., Schou, J., Nilsson, J., Choudhary, C. <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+Is+Not+Essential+for+Viability+of+Human+Cells+with+Genetically+Lowered+APC/C+Activity.">The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity.</a> Cell Rep. 1 (8), 1829-1840 (2016).</li><li>Iuso, D., 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=Exogenous+Expression+of+Human+Protamine+1+(hPrm1)+Remodels+Fibroblast+Nuclei+into+Spermatid-like+Structures.">Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures.</a> Cell Rep. 1 (9), 1765-1771 (2015).</li><li>Ratcliffe, E., Glen, K. E., Naing, M. W., Williams, D. 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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.
Thi...

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.

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			<td height="21" style="height:21px;width:191px;">DMEM/F12</td>
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		<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>
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		<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>
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			<td height="21" style="height:21px;width:191px;">Trypsin-EDTA</td>
			<td style="width:135px;">Gibco</td>
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			<td height="21" style="height:21px;width:191px;">Fetal bovine serum</td>
			<td style="width:135px;">Atlanta Biologicals</td>
			<td>S11150</td>
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			<td height="21" style="height:21px;">DMSO</td>
			<td style="width:135px;">EMD Millipore</td>
			<td>MX 1458-6</td>
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			<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>
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			<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>
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		<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>
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		<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>
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			<td height="21" style="height:21px;width:191px;">Nocodazole</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:128px;">M1404</td>
			<td></td>
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		<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>
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		<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>
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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 indi...

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 ...

The overall goal of this method is to label and visualize live chromosomes in mitotic cells using Histone 2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system. This method can be used to visualize mitotic chromosomes in cultured cells to study the effects of genetic modifications or drug treatments on chromosome segregation. The main advantage of this technique is that it provides an easy and convenient method to label and visualize live chromosomes in mitotic cells.Demonstrating the live imaging procedure will be Doctor Kazuyo Takeda who maintains the Microscopy and Imaging Core Facility in the Division of Viral Products at the FDA Center for Biologics Evaluation and Research. The mouse embryonic fibroblasts, or MEFs, used in this experiment are grown in a two-well chambered cover glass at 37 degrees Celsius and 5%CO2 overnight, as described in the text protocol. On the following day, synchronize the MEFs in G0/G1 phase by incubating in DMEM/F12 growth medium containing 0.1%FBS.On the day of labeling, prepare a 1.5 milligram per milliliter stock solution of nocodazole in DMSO. A critical step is to calculate the right amount of CL-HB particles per cells to achieve enough labeling for chromosome visualization without toxicity to cells. We have previously determined, by titration experiments, that 30 PPC works best.Add to the MEFs, 200 microliters of growth medium with 10%FCS, 200 nanograms per milliliter of nocodazole, and CL-HB reagent at 30 particles per cells. Incubate the MEFs at 37 degrees Celsius and 5%CO2 for 18 hours. Visualization of the MEFs is accomplished using a spinning disk confocal microscope system equipped with an environmental chamber and an oil immersion, 63X objective lens.One day before imaging, turn the environmental chamber power on and warm up the entire chamber at 37 degrees Celsius overnight. On the following day, turn the power on for the microscope stand, camera, spinning disk unit, illuminator, argon laser, computer, and motorized stage. After letting the system warm up for three minutes, start the argon laser by turning on the ignition key.Switch the toggle switch for the argon laser from standby to laser run. Launch the data acquisition and processing software. Initiate the CO2 controller for the stage top incubator, and set the concentration of CO2 at 5%Remove the chambered cover glass from the incubator, place it on the microscope stage, and select the oil immersion 63X objective lens.View through the ocular lenses, focus the image, and identify a cell in the stage of nuclear envelope breakdown, or NEBD. Initiate the 488-nanometer argon laser to visualize Histone 2B-GFP. Open the acquisition control window, and set exposure time for the GFP channel.Identify a cell that is in NEBD. Then, manually determine the cell's top and bottom focal plane, and enter the XYZ optical sectioning settings. Observe the cell for 20 minutes.It is important to be aware that since not all cells will go through mitosis, mitotic progress must be actively monitored. If the cell does not proceed through cell division after 20 minutes, stop image acquisition and move on to the next cell that is in NEBD. To obtain data for a movie, take pictures every three minutes.Approximately one hour per cell is needed to image lagging chromosomes during mitosis in PP2A-B56-gamma cells that escaped from the spindle assembly checkpoint. Save images in ZVI file format for further analysis. Live cell imaging was used to visualize the fate of individual cells as they progressed from NEBD through mitosis.Post-nocodazole treatment, all 21 wild-type cells observed were arrested at metaphase. In contrast, from the 21 cells lacking PP2A-B56-gamma observed, 16 were able to go through mitosis after nocodazole treatment. In addition, abnormal chromosomal segregation was detected in 13 of those MEFs.An example of a PP2A-B56-gamma cell undergoing chromosomal mis-segregation is shown in this video clip. Once mastered, this technique will take three to four days depending on the cell treatment prior to the labeling protocol. It is important to remember that this is a transient transfection and signals can be detected only up to five days.This protocol can be used to answer additional questions related to the cell cycle regulation, genomic instability, and the quality of the cell therapy products. After its development, this technique could be used to study live imaging of chromosomes in a wide variety of cell lines, such as primary cell lines, stem cells, neurons, and immortalized T-cells. After watching this video, you should have a good understanding of how to easily label and visualize live chromosomes in mitotic cells using Histone 2B-GFP BacMam 2 label and a spinning disc confocal microscopy system.

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