AFB, MAV and NJG would like to thank Ryan Quinton for comments on the manuscript and Adrian Salic for the TDRFP-NLS construct. AFB and MAV are funded by the NIGMS Biomolecular Pharmacology Training Grant 5T32GM008541. NJG is a member of the Shamim and Ashraf Dahod Breast Cancer Research Laboratories and is supported by NIH grants GM117150 and CA-154531, the Karin Grunebaum Foundation, the Smith Family Awards Program, the Searle Scholars Program, and the Melanoma Research Alliance.

This protocol focuses on using the non-transformed and chromosomally stable retinal pigmented epithelial (RPE-1) cell line for live-cell imaging experiments. However, this protocol can be adapted to any adherent cell line so long as cell culture conditions are adjusted as necessary. All procedures must adhere to institutional biosafety and ethical guidelines and regulations.
1. Preparing Cells for Live-cell Imaging
<ol>
	<li>Use freshly thawed and early passage RPE-1 cells expressing either human histone H2B fused to a fluorescent protein (e.g. H2B-GFP), or the FUCCI system (a detailed protocol on how to generate FUCCI-expressing cells can be found in reference45) to assess mitotic cell fate.
	<ol>
		<li>To measure the frequency of nuclear rupture, use RPE-1 cells expressing both H2B-GFP and a tandem dimer of red fluorescent protein fused to a single nuclear localization signal (TDRFP-NLS). 
		NOTE: Constructs of three green fluorescent proteins fused in tandem to a single nuclear localization signal (GFP3-NLS) have also been used to demonstrate rupture (as in reference42).</li>
		<li>Maintain cells on 10 cm tissue culture plates in the appropriate growth medium. RPE-1 cells are maintained in phenol red-free, Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM:F12) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 &#956;g/mL streptomycin.</li>
	</ol>
	</li>
	<li>Aspirate medium from the cells, wash the tissue culture dish with 10 mL of sterile, room temperature phosphate buffered saline (PBS) to remove remaining medium, and then aspirate the PBS.
	<ol>
		<li>Add 2 mL of 0.25% Trypsin with ethylenediaminetetraacetic acid (EDTA) to the cells and incubate at 37 &#176;C for 3 min or until the majority of cells have detached from the plate. 
		NOTE: Do not keep cells in trypsin longer than needed.</li>
	</ol>
	</li>
	<li>Add 10 mL of complete medium to collect the trypsinized cells with a 10 mL serological stripette and dispense in a 15 mL conical tube.
	<ol>
		<li>Pellet the cells by centrifugation at 180 x g for 3 min at room temperature.</li>
	</ol>
	</li>
	<li>Aspirate the supernatant, being careful not to disrupt the pellet, and then thoroughly resuspend the pellet in 10 mL of fresh medium by gently pipetting the medium up and down in the serological stripette.</li>
	<li>Use a hemocytometer or automated cell counting machine to count cells46.
	<ol>
		<li>Dilute cells in a new conical tube to 30,000 cells/mL of complete medium.</li>
		<li>Plate 30,000 RPE-1 cells (1 mL volume) per well of a 12-well glass bottomed (#1.5 thickness) imaging dish to achieve the required cellular density the next day (30&#8211;50% confluent). 
		NOTE: Always handle the plate with gloves and be careful not to touch the glass-bottom.</li>
	</ol>
	</li>
	<li>Allow cells to grow in a 37 &#176;C tissue culture incubator until they fully attach and flatten on the glass-bottomed imaging plate. While cells can attach in as little as 4 h, it is recommended that cells are not treated with drugs and imaged until the following day.</li>
	<li>Add paclitaxel (dissolved in dimethyl sulfoxide (DMSO)) to the desired final concentration in complete medium and mix thoroughly.
	<ol>
		<li>Prepare complete medium with an equal volume of DMSO alone to use as a control.</li>
		<li>Warm the medium containing drug or DMSO to 37 &#176;C before adding to cells. This will prevent focal drift due to a sudden temperature change.</li>
		<li>Add 1 mL of medium containing either paclitaxel or DMSO alone to individual wells of the 12-well imaging dish.</li>
	</ol>
	</li>
</ol>
2. Setting Up the Microscope for Live-cell Imaging
<ol>
	<li>Clean the glass-bottom of the imaging dish with optical cleaner to remove any fingerprints or dust that may interfere with imaging. Use sufficient optical cleaner to wet the entire glass surface.</li>
	<li>Place the glass-bottom dish on the microscope stage in the imaging dish adaptor, remove the plastic cover from the dish, and cover the dish with a glass-topped chamber. Ensure the chamber has a valve to allow a steady flow of air consisting of 5% CO2. 
	NOTE: To humidify the 5% CO2, the gas is flowed through tubing inserted into a sterile water bath housing. This allows for the gas to become equilibrated to 95% humidity.</li>
	<li>Initiate/calibrate the encoded stage using the software program controlling the microscope. This will ensure accurate X-Y coordinates and prevent focal drift.</li>
	<li>Focus on the cells using phase-contrast optics and perform Kohler illumination (described in detail in reference47) to focus the light and provide optimal contrast.</li>
	<li>Use acquisition software to determine the optimal exposure times for white light and all fluorescent channels being used (exposures that give 75% pixel saturation on the camera are ideal, provided this amount of light is not toxic to cells).
	<ol>
		<li>Use acquisition software to select several, non-overlapping fields of view from each well for imaging. Select imaging regions where cells have adhered well to the glass-bottom and are between 50% and 70% confluent. Avoid areas of clumped cells, as this will make subsequent analysis difficult. 
		NOTE: It is important to have non-overlapping fields of view from each well to avoid tracking the same cells twice. If cells are highly motile, it may be necessary to acquire several images radiating out from a central point and stitch them back together to make one large field of view.</li>
	</ol>
	</li>
	<li>Activate the microscope&#39;s autofocusing feature to ensure all points are maintained in focus for the duration of the experiment.</li>
	<li>To assess mitotic cell fate, set the imaging software to collect images from each selected field of view every 10 min for up to 96 h. Unperturbed mitosis lasts 20&#8211;40 min, and thus 10 min intervals will provide enough sampling to identify when cells divide. To identify nuclear rupture, which is a transient event, acquire images every 5 min. 
	NOTE: Make sure that the computer driving the image acquisition software has auto-updating, screensavers, and energy-savings modes disabled, as these can often interfere with image acquisition over long experiments.</li>
	<li>Initiate the imaging experiment. Periodically confirm that image acquisition is running smoothly over the course of the video.</li>
</ol>
3. Video Analysis to Identify Cell Fate in Response to Paclitaxel
<ol>
	<li>Confirm that imaged cells are healthy throughout the course of the imaging experiment. Control cells treated with DMSO should be viable and actively proliferating. 
	NOTE: If cells exhibit signs of stress, do not quantitate the video and instead focus on optimizing imaging conditions48. Signs of imaging stress include blebbing/dying cells, cells that fail to attach/spread on the plate, and cells that show a low mitotic index and/or prolonged mitosis. Possible sources of stress include fluctuations in temperature or CO2 levels inside the imaging chamber or phototoxicity48.</li>
	<li>When imaging an entire field of view with a 10X or 20X objective, hundreds of individual cells may be present. Therefore, to assist with quantitation, divide the field of view into smaller quadrants using available software tools. Score cells within each quadrant separately (as described in steps 3.3.1&#8211;3.6.2).</li>
	<li>When analyzing the video, track each cell in a merged view using phase-contrast and/or fluorescent images. Use software analysis tools to create the merged view.
	<ol>
		<li>Starting at the beginning of the video, identify a single interphase cell and track its progress through the cell cycle using phase optics. To track a single cell, observe the cell from frame to frame by eye. Identify interphase cells due to their flattened morphology (as assessed by phase-contrast) and their lack of DNA condensation (as assessed by H2B-GFP) (Figure 1A).</li>
	</ol>
	</li>
	<li>Identify cells that enter mitosis by observation of cell rounding (using phase-contrast optics) and/or chromosome condensation (using H2B-GFP) (Figure 1A-C). Both cell rounding and chromosome condensation are readily visualized by eye.
	<ol>
		<li>Annotate the time when the cell enters mitosis. Continue tracking the cell until it reaches its fate (anaphase as in step 3.5, cell death from mitosis as in step 3.6.1, or mitotic slippage as in step 3.6.2).</li>
	</ol>
	</li>
	<li>Control cells should efficiently align their chromosomes and enter anaphase within 1 h (Figure 1D). Visualize anaphase by phase-contrast optics as the cell begins to pinch into two or through visualization of poleward-moving chromosomes labeled with H2B-GFP (Figure 1A). Annotate the time when the cell undergoes anaphase.</li>
	<li>By contrast, cells treated with paclitaxel will remain rounded with condensed chromosomes for several hours (3-40 h) (Figure 1B-D).
	<ol>
		<li>Identify mitotically-arrested cells that undergo cell death. 
		NOTE: Cells that die during mitosis are visualized by phase-contrast microscopy, as cells will bleb, shrink, and/or rupture (Figure 1B). If imaging H2B-GFP, the chromosomes will also fragment during cell death.</li>
		<li>Identify mitotically-arrested cells that undergo cell slippage. 
		NOTE: Cells that undergo mitotic slippage are observed by phase-contrast microscopy, as they flatten back out into interphase and decondense chromosomes without undergoing anaphase (Figure 1C). Cells that undergo mitotic slippage give rise to large tetraploid cells that are often multinucleated (Figure 1C).</li>
	</ol>
	</li>
	<li>Continue tracking cells from the original field of view. Once a whole field of view is tracked, move to a separate field of view acquired from the same well and continue tracking cells.</li>
</ol>
4. Video Analysis to Identify Cell Fate Following Mitotic Slippage
<ol>
	<li>Assess cell fate following mitotic slippage (as described in step 3.6.2) using RPE-1 cells expressing the FUCCI system. In addition to phase-contrast imaging, it is necessary to acquire both red fluorescence (indicative of G1 phase) and green fluorescence (indicative of S/G2/M) images).
	<ol>
		<li>Track FUCCI RPE-1 cells as described in step 3.3 through 3.7.
		<ol>
			<li>To confirm that the FUCCI system is working properly, validate that control cells alternate expression of the red and green fluorescent proteins appropriately from analysis of the live-cell imaging data. Control cells should transition from exhibiting entirely nuclear red fluorescence to exhibiting entirely nuclear green fluorescence during interphase as cells progress from G1 to S phase. Cells should continue exhibiting green fluorescence throughout the completion of mitosis. Immediately following mitosis, cells should once again exhibit entirely red fluorescence during interphase. 
			NOTE: While methods exist to quantify both RFP and GFP fluorescence intensities from the FUCCI system in single cells using fluorescent traces49, this is often not necessary as the fluorescence color change is robust and visible by eye.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Identify cells arrested in mitosis using phase-contrast microscopy and track them until they undergo mitotic slippage, as described in 3.4 and 3.6.2. Cells that slip out of mitosis and back into interphase will change from exhibiting green fluorescence during mitosis to red fluorescence during G1 phase (Figure 2A-C).
	<ol>
		<li>Track these slipped cells using phase-contrast and epifluorescent imaging by eye to assess their cell fate (Figure 2D).
		<ol>
			<li>Identify cells that re-enter the cell cycle. These cells are identified by the red-to-green change in fluorescence expression using the FUCCI system that indicates G1/S transition (Figure 2A).</li>
			<li>Identify cells that undergo G1 cell cycle arrest. These cells are identified by expression of red fluorescence that persists for &#62;24 h (Figure 2B).</li>
			<li>Identify cells that die in interphase. These cells are identified by cell rupture/blebbing/shrinking using phase-contrast imaging (Figure 2C).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Video Analysis to Identify Frequency of Nuclear Envelope Rupture
<ol>
	<li>Use RPE-1 cells expressing H2B-GFP and TDRFP-NLS to assess nuclear envelope rupture. In addition to phase-contrast imaging, acquire both red fluorescence (TRITC) and green fluorescence (FITC) images. Acquire images every 5 min to visualize rupture. 
	NOTE: It is critical to generate cell lines in which the TDRFP-NLS is efficiently imported into the nucleus with minimal cytoplasmic fluorescence.</li>
	<li>Image cells as described in step 3.3 through 3.4. The TDRFP-NLS fluorescence signal and H2B-GFP should co-localize during interphase. Upon mitosis (as visualized by cell rounding using phase optics and/or chromosome condensation by H2B-GFP), the nuclear envelope will break down and the TDRFP-NLS signal will become cytoplasmic (Figure 3A). Following mitosis, the nuclear envelope will reform in the daughter cells and the TDRFP-NLS signal will become nuclear.</li>
	<li>Track daughter cells throughout the subsequent interphase by live-cell imaging. Identify nuclear rupture events by observing a transient burst of nuclear-localized TDRFP-NLS into the surrounding cytoplasm. Within minutes, the nuclear envelope will be repaired and the TDRFP-NLS will be relocalized to the nucleus (Figure 3A). 
	NOTE: Often, the nuclear DNA, as visualized by H2B-GFP, will be seen to protrude outside the rupture site as a small bleb.</li>
	<li>Score the fraction of nuclei that undergo a rupture event during interphase. To score this fraction, count the number of cells that undergo a rupture event over the total number of cells tracked.</li>
</ol>

The authors have nothing to disclose.

The most critical aspect of long-term live-cell imaging is ensuring the health of the cells being imaged. It is essential that cells be exposed to minimal extraneous environmental stressors, such as substandard conditions regarding temperature, humidity, and/or CO2 levels. It is also important to limit photodamage from fluorescent excitation illumination, as imaging stress is known to affect cell behavior50. Limiting photodamage can be achieved in multiple ways as detailed elsewhere<sup...

Anti-mitotic drugs have long been used in the chemotherapeutic regimens of various types of solid tumors and often show great efficacy1,2,3. Mechanistically, these drugs disrupt normal mitotic progression and promote mitotic arrest in rapidly proliferating cancer cells. However, cell fate in response to mitotic arrest is highly variable: while a fraction of cells undergoes cell death directly from mitosis, others exit out of mitosis and return to interphase as tetraploid cells (a process termed mitotic slippage)4,5,6,7,8. These interphase cells can execute apoptosis, undergo permanent cell cycle arrest, or even re-enter the cell cycle4,5,6,7,8,9,10,11. Cells that evade mitotic cell death by slipping into interphase, only to re-enter the cell cycle following drug removal, may therefore contribute to the re-emergence of cancer cell populations. Moreover, cells that slip from mitosis are tetraploid, and tetraploidy is known to promote chromosome instability that drives tumor relapse12,13,14,15. Defining the factors that control cell fate in response to anti-mitotic drug treatments is therefore critical to optimize current therapeutics.
In this protocol, we describe methods to directly observe and study the fate of cells that undergo prolonged mitotic arrest in response to the anti-mitotic drug paclitaxel. Paclitaxel is an established therapeutic in the clinic and has proven highly efficacious in many tumors types, including those of the breast, ovaries, and lungs16,17,18,19,20. Paclitaxel, which is a plant alkaloid derived from the bark of the Yew tree, stabilizes microtubules and thus prevents their dynamicity21,22. While dampening of microtubule dynamics by paclitaxel does not affect cell cycle progression from G1 through G2, the drug does lead to sustained activation of the spindle assembly checkpoint during mitosis by hindering kinetochore-microtubule attachment (reviewed in depth here23,24)25. As a consequence, anaphase onset is prevented in paclitaxel-treated cells and results in a prolonged mitotic arrest.
This protocol will first describe approaches to identify mitotic cells in live-cell imaging experiments. Mitosis can be visualized in adherent tissue culture cells due to two noticeable cell biological changes. First, chromosomes become highly condensed immediately prior to nuclear envelope breakdown. While often detectable by standard phase-contrast microscopy, chromosome condensation can be more clearly detected using fluorescent tags that label chromosomes (e.g. fluorescently-labeled histone proteins). Second, mitotic cells can also be identified by the dramatic morphological changes that result from cell rounding.
This protocol will then demonstrate how to use live-cell imaging approaches to track the fates of cells experiencing prolonged mitotic arrest. Cells arrested in mitosis undergo one of three distinct fates. First, cells can undergo cell death during mitosis. This phenomenon is readily visualized by light microscopy, as dying cells are observed to shrink, bleb, and/or rupture. Second, cells can exit from mitosis and return back to interphase without chromosome segregation or cytokinesis, a process termed mitotic slippage. The decondensation of chromosomes and/or the flattening of the mitotic cell readily identifies this process. Cells that slip from mitosis also often display irregular, multi-lobed nuclei and frequently harbor several micronuclei5. Third, cells arrested in mitosis can initiate anaphase and proceed through mitosis after a long delay. While uncommon at higher drug concentrations, this behavior suggests that the arrested cells may have satisfied the spindle assembly checkpoint, or that the spindle assembly checkpoint is partially weakened or defective. Anaphase onset can be visualized by chromosome segregation and subsequent cytokinesis using live-cell imaging.
Live-cell imaging methods to track the fate of cells that evade mitotic cell death by undergoing mitotic slippage will also be described. Cells that undergo mitotic slippage either die in the subsequent interphase, trigger a durable G1 cell cycle arrest, or re-enter the cell cycle to initiate a new round of cell division4. An approach using the FUCCI (fluorescent ubiquitination-based cell cycle indicator) system to determine the fraction of cells that re-enter the cell cycle following mitotic slippage will be described. FUCCI allows for the direct visualization of the G1/S transition and can be used in conjunction with long-term live-cell imaging both in vitro and in vivo26,27. The FUCCI system takes advantage of two fluorescently labeled proteins, truncated forms of hCdt1 (chromatin licensing and DNA replication factor 1) and hGeminin, whose levels oscillate based on cell cycle position. hCdt1 (fused to a red fluorescent protein) is present at high levels during G1 phase where it acts to license DNA for replication, but is ubiquitinated by the E3 ubiquitin ligase SCFSkp2 and degraded during S/G2/M phases to prevent re-replication of DNA26. By contrast, hGeminin (fused to a green fluorescent protein), is an inhibitor of hCdt1 whose levels peak during S/G2/M, but is ubiquitinated by the E3 ubiquitin ligase APCCdh1 and degraded at the end of mitosis and throughout G126. Consequently, FUCCI delivers a straightforward fluorescence readout of cell cycle phase, as cells exhibit red fluorescence during G1, and green fluorescence during S/G2/M. The FUCCI system is a significant advance over other approaches (such as bromodeoxyuridine staining) to identify proliferative cells, because it does not require cell fixation and allows for single cell imaging without the need for additional pharmacological treatments to synchronize cell populations. Though not discussed in this protocol, additional live-cell sensors have also been developed to visualize cell cycle progression, including a helicase B sensor for G128, DNA ligase-RFP29 and PCDNA-GFP30 sensors for S-phase, and the recent FUCCI-4 sensor, which detects all stages of the cell cycle31.
Finally, a live-cell imaging method to detect nuclear envelope rupture will be described. Recent studies have revealed that the nuclear envelopes of cancer cells are unstable and prone to bursting, thereby allowing the contents of the nucleoplasm and cytoplasm to intermix. This phenomenon, termed nuclear rupture, can promote DNA damage and stimulation of the innate immune response32,33,34,35,36,37,38,39,40,41,42,43,44. While the underlying causes of nuclear rupture remain incompletely characterized, it is known that deformations in nuclear structure correlate with an increased incidence of nuclear rupture42. One well-known effect of paclitaxel treatment is the generation of strikingly abnormal nuclear structures following mitosis; as such, a method using live-cell imaging to quantify nuclear rupture will be described, while also exploring if paclitaxel treatment increases the frequency of nuclear rupture events. Nuclear rupture can be detected by the observed leakage of a nuclear-targeted fluorescent protein into the cytoplasm (e.g. a tandem dimer repeat of RFP fused to a nuclear localization signal, TDRFP-NLS). This leakage is distinctly visible by eye, which enables simple quantitation of rupture events.
This protocol requires a widefield epifluorescence microscope that is equipped with an encoded stage and autofocusing software. The encoded stage allows for precise automated movement to defined X-Y coordinates, while autofocus software maintains cells in focus for the duration of the imaging period. In addition, this protocol requires equipment to maintain cells at 37 &#176;C with humidified 5% CO2 atmosphere. This can be achieved by enclosing the entire microscope within a temperature and atmosphere controlled enclosure, or by using stage-top devices that locally maintains temperature and environment. The phase-contrast objective used in this protocol is a plan fluor 10x with a numerical aperture of 0.30. However, 20X objectives are also sufficient to identify both rounded mitotic and flattened interphase cells in a single focal plane. If performing phase-contrast imaging (as described in this method), the cover can be either glass or plastic. If differential interference contrast (DIC) microscopy is used, it is imperative to use a glass cover to prevent depolarization of light.

<table><tbody><tr><td>phenol red-free, DMEM:F12 media</td><td>Hyclone</td><td>SH3027202</td><td></td></tr><tr><td>10% fetal bovine serum (FBS)</td><td>Gibco</td><td>10438026</td><td></td></tr><tr><td>100 IU/ml penicillin, and 100 mg/ml streptomycin.</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gemini</td><td>50-753-3104</td><td></td></tr><tr><td>12-well glass bottomed imaging dish</td><td>Cellvis</td><td>P12-1.5H-N</td><td></td></tr><tr><td>Paclitaxel</td><td>TSZ Chem</td><td>RS036</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma</td><td>NC0215085</td><td></td></tr><tr><td>Environmental Chamber</td><td>In Vivo Scientific</td><td></td><td></td></tr><tr><td>5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td>Z03NI7432000201</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Fisherbrand</td><td>05-539-12</td><td></td></tr><tr><td>10 cm polystyrene tissue culture plates</td><td>Falcon</td><td>08772E</td><td></td></tr><tr><td>10 mL Disposable Serological Pipette</td><td>Fisherbrand</td><td>4488</td><td></td></tr><tr><td>5804R 15 amp Centrifuge</td><td>Eppendorf</td><td>97007-370</td><td></td></tr><tr><td>1000 microliters Pipette</td><td>Gilson</td><td>Pipetman Classic</td><td></td></tr><tr><td>20 microliters Pipette</td><td>Gilson</td><td>Pipetman Classic</td><td></td></tr><tr><td>p1000 Pipette Tips</td><td>Sharp</td><td>p1126</td><td></td></tr><tr><td>p20 Pipette Tips</td><td>Sharp</td><td>P1121</td><td></td></tr><tr><td>RPE-1 Cells</td><td>ATCC</td><td>CRL-4000</td><td></td></tr><tr><td>Incubator Galaxy 170S</td><td>Eppendorf</td><td>CO17011005</td><td></td></tr><tr><td>Pipette Boy</td><td>Integra</td><td>156401</td><td></td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td>0267110</td><td></td></tr><tr><td>Nikon Ti-E-PFS Inverted Microscope for Live Cell Imaging</td><td>Micro Video Instruments, Inc.</td><td>MEA53100</td><td></td></tr><tr><td>Prior X-Y Stage System and White Light LED Illuminator</td><td>Micro Video Instruments, Inc.</td><td>500-H117P1N4</td><td></td></tr><tr><td>Prior Proscan 3 Controller XYZ with Encoders</td><td>Micro Video Instruments, Inc.</td><td>500-H31XYZE</td><td></td></tr><tr><td>Andor Digital Camera</td><td>Micro Video Instruments, Inc.</td><td>D10NLC</td><td></td></tr><tr><td>Nikon NIS Elements AR Software</td><td>Micro Video Instruments, Inc.</td><td>MQS31000</td><td></td></tr><tr><td>SOLA LED Illuminator for Fluorescence</td><td>Micro Video Instruments, Inc.</td><td>SOLA-E</td><td></td></tr><tr><td>InVivo Environmental Chamber with CO2 Flow Control</td><td>Micro Video Instruments, Inc.</td><td>Ti-IVS-100</td><td></td></tr><tr><td>Ti-ND6-PFS Perfect Focus Motorized Nosepiece</td><td>Micro Video Instruments, Inc.</td><td>MEP59391</td><td></td></tr><tr><td>Ti-C System Condenser Turret</td><td>Micro Video Instruments, Inc.</td><td>MEL51000</td><td></td></tr><tr><td>Ti-C LWD LWD Lens Unit for System Condenser Turret</td><td>Micro Video Instruments, Inc.</td><td>MEL56200</td><td></td></tr><tr><td>Te-C LWD Ph1 Module</td><td>Micro Video Instruments, Inc.</td><td>MEH41100</td><td></td></tr><tr><td>CFI Plan Fluor DLL 10X Objectivena 0.3 wd 16mm</td><td>Micro Video Instruments, Inc.</td><td>MRH10101</td><td></td></tr><tr><td>CFI Super Plan Fluor ELWD 20xc ADM Objective</td><td>Micro Video Instruments, Inc.</td><td>MRH48230</td><td></td></tr></tbody></table>

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.

Using the protocol described above, RPE-1 cells expressing H2B-GFP were treated with an anti-mitotic or vehicle control (DMSO) and analyzed by live-cell imaging. Analysis revealed that 100% of control mitotic RPE-1 cells initiated anaphase an average of 22 min after entering mitosis (Figure 1A, 1D). By contrast, RPE-1 cells treated with paclitaxel exhibited a profound mitotic arrest lasting several hours (Figure 1D</stron...

Watch this Scientific Journal Video about Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel at JoVE.com

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

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 ...

long-term-live-cell-imaging-to-assess-cell-fate-response-to

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JoVE Journal

Cancer Research

9.9K Views.  Boston University School of Medicine. Live-cel imaging biedt een schat aan informatie op afzonderlijke cellen of hele populaties die onbereikbaar is door de vaste cel imaging alleen. Hier, worden live-cel imaging protocollen te beoordelen cel lot beslissingen naar aanleiding van de behandeling met de anti-mitotische drug paclitaxel beschreven.

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

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Medicine

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

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 ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

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

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