DLM is supported by an NSF GRFP. ALM is supported by funding from the Smith Family Award for Excellence in Biomedical Research.

1. Generation of hTERT-RPE-1 cells stably expressing RFP-Histone 2B (RFP-H2B) and &#945;-tubulin-EGFP (tub-EGFP)
NOTE: All steps follow aseptic techniques and take place in a biosafety level II+ (BSL2+) safety cabinet.
<ol>
	<li>Generate retrovirus carrying the genes of interest (&#945;-tubulin-EGFP and RFP-H2B) by the transfection of 293T cells with the appropriate lentiviral plasmids according to the manufacturer&#39;s instructions of the lipid-based transfection delivery system.
	<ol>
		<li>Day 1: Use a disposable glass Pasteur pipette to aspirate the cell culture medium from a plate of sub-confluent 293T cells and wash off the residual medium with phosphate buffered saline (PBS) by adding 5 mL of PBS. Swirl to distribute PBS over the plate bottom, then aspirate the PBS with a sterile disposable glass Pasteur pipette.</li>
		<li>Add 2 mL of 0.05% trypsin that has been pre-warmed to 37 &#176;C and return the plate to a humidified incubator at 37 &#176;C with 5% CO2 for 2 to 5 min to allow adherent cells to be released from the cell culture dish surface.</li>
		<li>Add 8 mL of fresh Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin to the plate containing trypsin.</li>
		<li>Resuspend the cells by gently pipetting and transfer the suspension to a sterile 15 mL conical tube. Place the conical tube in a balanced centrifuge and spin at 161 x g for 5 min at room temperature to gently pellet the cells.</li>
		<li>Aspirate the medium/trypsin solution from pelleted cells and resuspend cells in 10 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Use a hemocytometer to count the 293T cell suspension and plate 2 x 106 293T cells per well of a 6 well plate.</li>
		<li>Culture cells in a total volume of 2 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintain&#160;in a humidified incubator at 37 &#176;C with 5% CO2.</li>
		<li>Day 2: Pipette 7 &#181;L of lipid-based transfection delivery reagent and 100 &#181;L of reduced serum medium into a 1.5 mL microcentrifuge tube and allow it to incubate at room temperature for 5 min (tube #1).</li>
		<li>In a separate 1.5 mL microcentrifuge tube, pipette 1 &#181;g of RFP-H2B expression vector (or 1 &#181;g of &#945;-tubulin-EGFP expression vector), together with 5 &#181;L enhancer reagent (as required per manufacturer&#39;s guidelines), 0.5 &#181;g pMD2.G, and 1 &#181;g psPAX2 and 100 &#181;L of reduced serum medium (tube #2).</li>
		<li>Combine the contents of step 1.1.7 and step 1.1.8 by carefully pipetting tube #2 into tube #1 and incubate for 20 min at room temperature. 
		NOTE: The reaction can be scaled as needed for transfection of cells in additional wells.</li>
		<li>Pipette the transfection reaction dropwise to the desired well containing 293T cells in 2 mL of medium and return the dish to the humidified incubator at 37 &#176;C with 5% CO2. 
		NOTE: To generate cells expressing both RFP-H2B and &#945;-tubulin-EGFP, generate separate virus for each expression construct (steps 1.1.7- 1.1.9).</li>
		<li>Day 3: Aspirate and replace the medium with 2 mL of fresh DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Return the dish to the humidified incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Day 4: Collect the medium containing expressed virus particles, being cautious not to disrupt or remove 293T cells. Filter the medium containing virus particles by passing it through a 0.45 &#181;M filter attached to a 5 mL syringe. Aliquot virus for short term storage at 4 &#176;C, or long term storage at -80 &#176;C. 
	NOTE: Viral particles obtained in this manner will be present in a range of ~1 x 107 to 1 x 108 Transducing Units/mL. As the viral-producing cells and cells to be infected are both cultured in the same medium, viral concentration to replace the medium is not required and filtered viral particles can be used directly to infect cells.</li>
	<li>Seed 2 x 105 hTERT-RPE-1 cells per well of a 6-well dish in preparation for the viral infection. Culture cells in 2 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and maintain in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>Day 5: Add hexadimethrine bromide (e.g., polybrene)to the hTERT-RPE-1 cells to a final concentration of 8 &#181;g/mL. Infect cells with a mixture of 500 &#181;L of virus diluted in 500 &#181;L of cell culture medium. 
	NOTE: Hexadimethrine bromide stock solution is prepared in sterile distilled water, then filtered through a 0.45 &#956;m filter.</li>
	<li>Day 6: Using a disposable glass Pasteur pipette, aspirate the medium from wells containing the virus infected cells. Replace the cell culture medium with 2 mL of DMEM containing 10% FBS, 1% penicillin/streptomycin, and appropriate concentrations of antibiotic to select for the plasmid integration and expression. 
	NOTE: The &#945;-tubulin-EGFP expression plasmid used in the described experiments carries a puromycin-resistance gene and the RFP-H2B expression plasmid carries a blasticidin resistance gene. Therefore, 10 &#181;g/mL of puromycin and 2 &#181;g/mL of blasticidin are used for the selection of &#945;-tubulin-EGFP, RFP-H2B expressing hTERT RPE-1 cells. Concentrations of antibiotic used for the selection may differ for various cell lines used.</li>
	<li>Maintain cells under antibiotic selection for 5-7 days, replacing the medium every 3 days with fresh medium containing appropriate selection reagents. 
	NOTE: Cells should be maintained at sub-confluence during the selection and should be expanded as needed, as described in steps 1.1.1 to 1.1.4.</li>
	<li>Use immunofluorescence imaging to confirm the expression of tagged constructs. 
	NOTE: If desired, single cell clones can be derived to obtain uniform expression levels within the cell population. Once stable clones are confirmed, cells can be maintained under the standard culture conditions in the absence of antibiotic selection.</li>
</ol>
2. Preparation of cells for live cell imaging following mitotic spindle perturbations
NOTE: Use aseptic techniques and perform the steps in a BSL2 safety cabinet.
<ol>
	<li>Using a sterile disposable glass Pasteur pipette, aspirate the medium from the culture plate containing the cell line that carries the expression construct(s) (from step 1.7). Briefly wash the cells with 10 mL of sterile PBS. Swirl to distribute PBS over the plate bottom, then aspirate PBS with a sterile disposable glass Pasteur pipette.</li>
	<li>Add 2 mL of 0.05% trypsin to the 10 cm plate. Incubate the plate at 37 &#176;C for 2-5 min or until cells have detached from the plate surface.</li>
	<li>Add 8 mL of fresh medium to the plate containing trypsin. Resuspend the cells by gently pipetting and transfer the suspension to a sterile 15 mL conical tube. Place the conical tube in a balanced centrifuge and spin at 161 x g for 5 min at room temperature to gently pellet the cells.</li>
	<li>Carefully aspirate the supernatant and resuspend in 10 mL of PBS by pipetting gently with a 10 mL serological pipette. Place the conical tube in a balanced centrifuge and spin at 161 x g for 5 min at room temperature to gently pellet the cells.</li>
	<li>Carefully aspirate the supernatant and resuspend in 10 mL of the fresh medium by pipetting gently with a 10 mL serological pipette.</li>
	<li>Count cells, then calculate the cell number using a hemacytometer and dilute to a concentration of 1-2 x 105 cells/mL in the cell culture medium. Seed 500 &#181;L of the cell suspension to each well of a sterile 12-well imaging bottom plate. Place the plate in the cell culture incubator and allow cells to adhere to the plate surface. 
	NOTE: Live cell imaging requires cells to be well-adhered so that they remain associated with the imaging plane during image acquisition. The duration of the time needed for cells to adhere following plating can differ from one cell line to the next and should be optimized for the cell line under study.</li>
	<li>Up to 30 min prior to initiating time-lapse imaging, add a relevant concentration of a mitotic drug to one or more of the wells seeded with cells. To account for the potential impact of the organic solvent on cellular behavior, add an equal volume of the inhibitor&#39;s diluent to cells as controls. For example, ensure that the addition of 100 nM of the specific inhibitor of the mitotic kinase Aurora A (e.g., alisertib) is paralleled with an equal volume of DMSO, the diluent for this drug, in a control well of cells. 
	NOTE: Comparative analysis of dynamic changes in the mitotic progression require wells of cells to be prepared in the absence of perturbations to enable imaging of normal mitoses in parallel to experimental conditions.</li>
</ol>
3. Microscope set up for time-lapse imaging of RFP-H2B, &#945;-tubulin-GFP expressing cells (Figure 1, Figure 2)
<ol>
	<li>Place the cell culture plate containing RFP-H2B, &#945;-tubulin-GFP expressing hTERT RPE-1 cells to be imaged into an appropriate stage insert on an inverted epifluorescence microscope that is equipped with a high resolution camera (pixel size of 0.67 &#956;m at 20x), an environmental chamber preheated to 37 &#176;C, and a delivery system for humidified 5% CO2. 
	NOTE: Either enclosed environmental chambers or temperature controlled stage inserts may be appropriate provided humidified 5% CO2 can be delivered and stable temperature control obtained.</li>
	<li>Use a 20x air objective with a numerical aperture of 0.5 and equipped for the high contrast fluorescence and phase contrast or brightfield imaging. View cells with the phase contrast or brightfield and adjust the course and fine focus on the microscope to bring cells into focus.</li>
	<li>Identify and set the optimal exposure times for brightfield, GFP, and RFP image acquisition by selecting the respective filter cube with appropriate excitation and emission for the fluorophores that will be imaged. Alternatively, click the auto exposure button to input&#160;a predetermined exposure time. If the signal is not sufficiently intense, select pixel binning to enable shorter exposure times by clicking on the binning tab and selecting 2 x 2 pixel binning from the drop down menu. 
	NOTE: Phototoxicity can impair mitotic progression and cell viability. Failure of mitotic cells in control populations to complete normal mitoses may be an indication that exposure times and/or imaging duration needs to be further optimized.</li>
	<li>Use an acquisition and analysis software that enables multi-coordinate, multi-well imaging to be acquired concurrently and define the parameters for the image acquisition.
	<ol>
		<li>Select and calibrate the microscope stage to the multi-well dish, according to the manufacturer&#39;s instructions. Use the image acquisition software to highlight or otherwise select the wells that will be imaged by clicking on the relevant wells in the diagram of the plate format that is being used.</li>
		<li>Under the GeneratedPoints control panel (Figure 2), define the coordinates within each well that will be imaged by clicking on the Point Placement tab and selecting predefined or random coordinate placement from the drop down menu. Select the Working Area tab and select restricted from the drop down menu to restrict the coordinate selection area to exclude the boundaries of the well. Click the Count and Distribution tabs to select the number and distribution of points to be captured per well, respectively. 
		NOTE: The number of coordinates that can be imaged per well within the 5 min timepoint increment will be limited by the number of wells to be imaged, as well as the exposure time for each channel. Typically, 5-8 coordinates per well are sufficient to observe at least 50 cells in each condition progress through mitosis within 4 h.</li>
		<li>Select and input the time interval and duration to collect images by clicking on and inputting the values in the TimeSequence control panel. 
		NOTE: Acquisition of images every 1 to 5 minutes is appropriate to monitor the dynamics of mitotic progression. The overall&#160;duration of imaging should reflect the desired endpoint and proliferation rate of the cell line. For example, 4 hours is sufficient to see many cells progress through mitosis, 16 hours is sufficient to see most RPE-1 cells in an asynchronous population progress through mitosis.</li>
	</ol>
	</li>
</ol>
4. Time-lapse image analysis to determine metaphase timing and mitotic cell fate following mitotic spindle perturbations
NOTE: Perform the image analysis using an image acquisition software (Table of Materials), ImageJ, or comparable image analysis software.
<ol>
	<li>Visualize RFP-H2B labeled chromatin by selecting the images captured with the RFP filter cube in place. Identify a cell entering mitosis as indicated by initial chromatin compaction (Figure 2C, blue arrowhead) and nuclear envelope break down (when &#945;-tubulin-EGFP is no longer excluded by the nuclear boundary).</li>
	<li>Determine the mitotic timing of metaphase alignment and anaphase onset in individual cells: Track the cell through consecutive timepoints in the acquired movie to determine the number of timepoints/minutes from mitotic entry until RFP-H2B-labelled chromatin completes alignment at the cell equator during metaphase (Figure 2C, yellow arrowhead).</li>
	<li>To monitor mitotic timing, mitotic fidelity, and cell fate, continue to track the cell through consecutive timepoints to identify the time coordinate at which anaphase chromosome segregation is apparent (Figure 2C, white arrowhead) and/or where chromatin decompaction and nuclear envelope reformation (as indicated by tubulin exclusion from the nucleus) has occurred. 
	NOTE: Perturbations in spindle assembly or mitotic progression can be assessed as a function of the time required to obtain a bipolar mitotic spindle and achieve complete chromosome alignment, or to complete anaphase chromosome segregation.</li>
	<li>Visualize RFP-H2B to identify cells in each population that exhibit mitotic defects including lagging chromosomes and chromatin bridges during anaphase chromosome segregation. 
	NOTE: Compromised mitotic fidelity may also result in multinucleated or micronucleated daughter cells and can be visualized in cells post mitotic exit, as in Figure 3.</li>
</ol>

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The authors have nothing to disclose.

The temporal resolution provided by the time-lapse imaging allows for the visualization and assessment of sequential cellular events within single cells. Approaches that make use of cellular synchronization followed by the collection and fixation of cells at sequential time points are limited in that comparisons are ultimately made between populations of cells. In contexts where the cellular response to perturbations may be non-uniform, or where the process being visualized is dynamic, live cell time-lapse imaging is bet...

Image-based analysis of fixed cells is commonly used to assess the cell population level changes in response to various perturbations. When combined with cell synchronization, followed by the collection and imaging of serial time points, such approaches can be used to suggest a cellular sequence of events. Nevertheless, fixed cell imaging is limited in that temporal relationships are implied for a population and not demonstrated at the level of individual cells. In this way, while fixed cell imaging and analysis is sufficient to observe robust phenotypes and steady-state changes, the ability to detect transient changes over time and changes that impact only a subpopulation of the cells is imperfect. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular and subcellular processes within a single cell, or cellular population, over time and without the aid of synchronization approaches that may themselves impact cellular behavior1,2,3,4,5,6.
The formation of a bipolar mitotic spindle is essential for the proper chromosome segregation during cell division, resulting in two genetically identical daughter cells. Defects in mitotic spindle structure that corrupt mitotic progression and compromise the fidelity of chromosome segregation can result in catastrophic cell divisions and reduced cell viability. For this reason, mitotic poisons that alter spindle formation are promising therapeutics to limit the rapid proliferation of cancer cells7,8,9. Nevertheless, fixed cell analysis of spindle structure following the addition of mitotic poisons is limited in its ability to assess the dynamic process of spindle formation and may not indicate whether observed changes in spindle structure are permanent or are instead transient and may be overcome to permit successful cell division.
In this protocol, we describe an approach to assess the dynamics of mitosis following spindle perturbations by live cell imaging. Using the hTERT immortalized RPE-1 cell line engineered to express an RFP-tagged Histone 2B to visualize chromatin, together with an EGFP-tagged &#945;-tubulin to visualize microtubules, the timing of metaphase chromosome alignment, anaphase onset, and ultimately mitotic cell fate are assessed using visual cues of chromosome movement, compaction, and nuclear morphology.

<table><tbody><tr><td>0.05% Trypsin</td><td>Gibo-Life sciences</td><td>25-510</td><td>A serine protease used to release adherent cells from culture dishes</td></tr><tr><td>15ml centrifuge tubes</td><td>Olympus Plastics</td><td>28-101</td><td></td></tr><tr><td>20x CFI Plan Fluor objective&amp;nbsp;</td><td>Nikon</td><td></td><td>For use in Live-cell imaging to visualize both bright field and fluorescence</td></tr><tr><td>293T Cells</td><td>ATCC</td><td>CRL-3216</td><td>For use in retroviral transfection; used in step 1.1</td></tr><tr><td>a-tubulin-EGFP&amp;nbsp;</td><td>Addgene</td><td>various numbers</td><td>Expression vector for alpha tubulin fused to a green fluorescent protein tag; for use in the visualization of tubulin in live-cell imaging: commercially available through addgene and other vendors</td></tr><tr><td>Alisertib</td><td>Selleckchem</td><td>S1133</td><td>Small molecule inhibitor of the mitotic kinase Aurora A. Stock concentration is prepared at 10mM in DMSO, and used at a final concentration of 100nM.</td></tr><tr><td>Blasticidin</td><td>Invitrogen</td><td>A11139-03</td><td>Antibiotic selection agent; used to select for a-tub-EGFP expressing cells</td></tr><tr><td>C02</td><td>Airgas</td><td></td><td>For use in cell culture and live cell imaging</td></tr><tr><td>Chroma ET-DS Red (TRITC/Cy3)</td><td>Chroma</td><td>49005</td><td>Single band filter set; excitation wavelength 545nm with 25nm bandwidth and emission at 605nm wavelength with 70nm bandwidth; for visualization of H2B-RFP</td></tr><tr><td>Chroma ET-EGFP (FITC/Cy2)</td><td>Chroma</td><td>49002</td><td>Single band filter set; excitation wavelength 470nm with 40nm bandwidth and emission at 525nm wavelength with 50nm bandwidth; for visualization of GFP-tubulin</td></tr><tr><td>disposable glass Pastuer pipets, sterilized&amp;nbsp;</td><td>Fisher Scientific</td><td>13-678-6A</td><td>For use in aspirating cells&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Gibo-Life sciences</td><td>11965-084</td><td>Cell culture medium for growth of RPE-1 and 293T cells</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibo-Life sciences</td><td>10438-026</td><td>Cell culture medium supplement</td></tr><tr><td>Lipofectamine 3000 and p3000</td><td>Invitrogen</td><td>L3000-015</td><td>Lipid based transfection reagent for transfection of plasmids; used in 1.1.4</td></tr><tr><td>Multi well Tissue Culture dishes</td><td>Corning</td><td>various</td><td>for use in cell culture, transfection/infection, and live cell imaging</td></tr><tr><td>Nikon Ti-E microscope</td><td>Nikon</td><td></td><td>Inverted epifluorescence microscope for use in live-cell imaging</td></tr><tr><td>NIS Elements HC&amp;nbsp;</td><td>Nikon</td><td>Version 4.51</td><td>Image acquisition and analysis software; used in sections 3 &amp;amp; 4</td></tr><tr><td>OPTI-MEM</td><td>Gibo-Life sciences</td><td>31985-070</td><td>Reduced serum medium for cell transfection; used in step 1.1.3</td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>Gibo-Life sciences</td><td>15140-122</td><td>antibiotic used in cell culture medium</td></tr><tr><td>phosphate bufferred saline (PBS)</td><td>Caisson labs</td><td>PBP06-10X1LT</td><td>sterile saline solution for use with cell culture</td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td>Lentiviral VSV-G envelope expression construct; used in step 1.1.4</td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich</td><td>H9268</td><td>Cationic polymer used to enhane viral infection efficiency; used in step 1.1.10</td></tr><tr><td>psPAX</td><td>Addgene</td><td>12260</td><td>2nd generation lentiviral packaging plasmid; used in step 1.1.4</td></tr><tr><td>Puromycin</td><td>Invitrogen</td><td>ant-pr-1</td><td>Antibiotic selection agent; used to select for RFP-H2B expressing cells</td></tr><tr><td>RFP- Histone 2B (H2B)</td><td>Addgene</td><td>various numbers</td><td>Expression vector for red fluorescent protein-tagged histone 2B; for use in the visualization of chromatin in live-cell imaging: commercially available through Addgene and other vendors</td></tr><tr><td>RNAi Max</td><td>Invitrogen</td><td>13778-150</td><td>Lipid based transfection reagent for transfection of siRNA constructs</td></tr><tr><td>RPE-1 cells</td><td>ATCC</td><td>CRL-4000</td><td>Human retinal pigment epithelial cell line</td></tr><tr><td>Tissue culture dish 100x20mm</td><td>Corning&amp;nbsp;</td><td>353003</td><td>for use in culturing adherent cells</td></tr><tr><td>Zyla sCMOS camera&amp;nbsp;</td><td>Nikon</td><td></td><td>Camera attached to the micrscope, used for capturing images of cells</td></tr></tbody></table>

live cell imaging to assess the dynamics of metaphase timing and cell fate following mitotic spindle perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Assessment of mitotic progression in the presence of spindle perturbations 
The regulation of spindle pole focusing is an essential step in proper bipolar spindle formation. Disruption in this process through protein depletions, drug inhibition, or alterations in centrosome number corrupt spindle structure and delay or halt mitotic progression10,11,12,<sup class="...

Watch this Scientific Journal Video about Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations at JoVE.com

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

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8.1K Views.  Worcester Polytechnic Institute. Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Video: Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Cancer Research

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

Genetics

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.

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 ...

Developmental Biology

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Neuroscience

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Medicine

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

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Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Live Cell Imaging of Chromosome Segregation During Mitosis

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Time-lapse Imaging of Mitosis After siRNA Transfection

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles