We thank the members of the Jha laboratory for their helpful discussion and comments. S.J. was supported by grants from the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centers of Excellence initiative to the Cancer Science Institute of Singapore (R-713-006-014-271), the National Medical Research Council (CBRG-NIG; BNIG11nov001 and CS-IRG; R-713-000-162-511), the Ministry of Education Academic Research Fund (MOE AcRF Tier 1 T1-2012 Oct -04). Y.Z., G.S.C., and C.Y.T. were supported by a post-graduate fellowship awarded by the Cancer Science Institute of Singapore, National University of Singapore.

1. Preparation
<ol>
	<li>Prepare MCF10A culture medium: Dulbecco&#39;s Modified Eagle Medium (DMEM)/F12 (1:1) with 5% horse serum, 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 &#181;g/mL insulin.</li>
	<li>Prepare serum-free medium: DMEM/F12 (1:1) with 20 ng/mL epithelial growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 &#181;g/mL insulin.</li>
	<li>Use the following siRNA sequence: 
	siControl: 
	Forward: 5&#39;-CGUACGCGGAAUACUUCGAdTdT-3&#39; 
	Reverse: 5&#39;-UCGAAGUAUUCCGCGUACGdTdT-3&#39; 
	siTIP60: 
	Forward: 5&#39;-UGAUCGAGUUCAGCUAUGAdTdT-3&#39; 
	Reverse: 5&#39;-UCAUAGCUGAACUCGAUCAdTdT-3&#39;</li>
	<li>Use 10-cm cell culture dishes and 24-well plates.</li>
</ol>
2. Transient Depletion of TIP60 Using siRNA
<ol>
	<li>On day 1, aliquot 15 &#181;L of transfection reagent (e.g., Lipofectamine RNAiMAX) and 2 mL of reduced serum medium (e.g., Opti-MEM) and mix them with 10 &#181;L of each siRNA (10 &#181;M stock), respectively.</li>
	<li>Incubate the mixture at room temperature for 20 min.</li>
	<li>Seed 1 x 106 MCF10A cells together with 3 mL of culture medium in a 10 cm dish. Count the cells using a hemocytometer.</li>
	<li>Add the siRNA mixture drop by drop to the 10 cm dish.</li>
	<li>Shake the dish to ensure that the cells are equally distributed before putting it into the 37 &#176;C incubator.</li>
	<li>After 6 h of incubation, replace the siRNA-containing medium with 8 mL of fresh culture medium.</li>
	<li>On day 2, prepare another batch of siRNA mixtures and incubate them at room temperature for 20 min.</li>
	<li>Aspirate out 8 mL of the culture medium added on day 1 and replace it with 3 mL of fresh culture medium.</li>
	<li>Add the second batch of siRNA mixture to the dishes drop by drop. Shake the dishes to ensure that the reagents are fully mixed before putting them into the incubator.</li>
	<li>After 6 h, replace the siRNA-containing medium with 8 mL of fresh culture medium.</li>
</ol>
3. Seed Cells for the Wound-healing Assay
<ol>
	<li>On day 3, discard the medium and wash the cells once with 2 mL of phosphate-buffered saline (PBS) buffer. Discard the PBS buffer.</li>
	<li>Trypsinize the siControl and siTIP60-treated cells by adding 1 mL of 1x&#160;trypsin-EDTA buffer to the plate. Put it back in the incubator for 20 min.</li>
	<li>Add 4 mL of complete culture medium to each plate, resuspend the cells by pipetting, and transfer them to a 15-mL tube.</li>
	<li>Centrifuge at 200 g for 5 min and remove the supernatant.</li>
	<li>Re-suspend the cells with 5 mL of serum-free medium and count the cells using a hemocytometer.</li>
	<li>Prepare a cell suspension of 6 x 105 cells/mL for both the siControl and siTIP60; ensure that the cells are well-mixed. Add 500 &#181;L of the cell suspension to one well of a 24-well plate to ensure 100% confluency. Seed 5 to 6 wells each for siControl and siTIP60.</li>
	<li>Gently shake the plate back and forth and then side-to-side to ensure even distribution. Avoid a circular motion. Leave the plate in the incubator for 24 h.</li>
	<li>Harvest the remaining cells to check the knockdown efficiency of TIP60. To do so, centrifuge the remaining cell suspension at 200 g for 5 min and remove the supernatant.</li>
	<li>Isolate RNA using a commercial reagent, following the manufacturer&#39;s protocol, and proceed to real-time quantitative PCR to evaluate the knockdown efficiency.</li>
</ol>
4. Create the Wound
<ol>
	<li>Check under a phase contrast microscope to ensure that the cells are fully attached. Observe the cells under the microscope (4X, 10X, and 20X&#160;magnification) to ensure that the cells are fully attached that a confluent monolayer of cells has formed. 
	NOTE: siControl cells usually attach better than siTIP60 cells. This is expected because the depletion of TIP60 makes the cells more mesenchymal-like20. Here, a normal benchtop white-light phase contrast microscope is used.</li>
	<li>To generate the wound, gently scratch a straight line across the center of the well with a 200 &#181;L pipette tip; the length of the wound generated is same as the diameter of the well. Repeat this step for all remaining wells. Use a new pipette tip for each well.</li>
	<li>Gently wash each well 5 times using serum-free medium to remove the detached cells.</li>
	<li>Add 1 mL of serum-free medium to each well and proceed to live-cell imaging.</li>
</ol>
5. Setup of Live-cell Imaging
<ol>
	<li>Turn on the microscope and the temperature control at least 1 h before setting up live-cell imaging to ensure that the temperature in the chamber reaches 37 &#176;C.</li>
	<li>Turn on the carbon dioxide (CO2) gas supply before starting the live-cell imaging. Set the CO2 concentration to 5%.</li>
	<li>Place the 24-well plate on the stage of the microscope and select the objective lens with 10X&#160;magnification for imaging. Direct the emitted light to the eyepieces instead of the camera and adjust the focus to locate the wound and the cells around it. 
	NOTE: In this case, white light with phase contrast was used.</li>
	<li>Use a live-cell observer system to mark the position of the wound in each well. 
	NOTE: The automated stage of the live-cell imaging system allows for image acquisition at multiple positions. Images are taken on every marked position once every interval.</li>
	<li>Set up the time condition: 1 h interval and 72 h duration; according to this setting, the system will take pictures at each marked position every hour for 72 h. Set up any desired time interval and period for monitoring. 
	NOTE: Here, a duration of 72 h is best because these cells start dying after 72 h in serum-free medium.</li>
</ol>
6. Tracking Single-cell Movement
<ol>
	<li>Export the data after 72 h. Export the data both as images in JPEG format and as videos in AVI format. 
	NOTE: This is important because ImageJ software can only recognize videos in AVI format. The different cell migration speeds between siTIP60 and siControl cells can be clearly observed in the video.</li>
	<li>Open the AVI video in the ImageJ software.</li>
	<li>Open the MTrackJ plugin (the plugin must be downloaded and installed separately): Plugins &#62; MtrackJ.</li>
	<li>Add a track for single cells chosen from the video by clicking the &#34;Add&#34; tab in the MtrackJ toolbar. Choose a cell at the tip of the invasive front and a cell that can migrate on its own (single-cell migration). Follow the movement of the chosen cells on the video and trace them using MtrackJ.</li>
	<li>Measure the distance of movement by clicking the &#34;measure&#34; tab on the toolbar. 
	NOTE: Here, almost no cells in the siControl moved as single cells, making it difficult to measure single-cell movement, whereas many of the siTIP60 cells moved as single cells. However, the movement of cells located at the invasive front can still be traced and calculated in both siControl and siTIP60 cells.</li>
	<li>Save the video with the tracks in AVI format.</li>
</ol>
7. Quantification of Cell Migration Ability
<ol>
	<li>For quantification, select images at 0 h and images at &#34;n&#34; h, where &#34;n&#34; refers to the timepoint when either the siTIP60 or siControl cells have completely closed the wound. 
	NOTE: Here, 48 h was chosen, as the wounds in siTIP60 cells were almost completely closed.</li>
	<li>Quantify the percent migration using ImageJ using the following formula: (area of wound at 0 h - area of wound at &#34;n&#34; h) / area of wound at 0 h x 100.</li>
</ol>

<ol>
	<li>Sun, Y., Jiang, X., Chen, S., Fernandes, N., Price, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+Tip60+histone+acetyltransferase+in+the+acetylation+and+activation+of+ATM.">A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM.</a> Proc Natl Acad Sci U S A. 102 (37), 13182-13187 (2005).</li><li>Sykes, S. M., Stanek, T. J., Frank, A., Murphy, M. E., McMahon, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+of+the+DNA+binding+domain+regulates+transcription-independent+apoptosis+by+p53.">Acetylation of the DNA binding domain regulates transcription-independent apoptosis by p53.</a> J Biol Chem. 284 (30), 20197-20205 (2009).</li><li>Jha, S., Shibata, E., Dutta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Rvb1/Tip49+is+required+for+the+histone+acetyltransferase+activity+of+Tip60/NuA4+and+for+the+downregulation+of+phosphorylation+on+H2AX+after+DNA+damage.">Human Rvb1/Tip49 is required for the histone acetyltransferase activity of Tip60/NuA4 and for the downregulation of phosphorylation on H2AX after DNA damage.</a> Mol Cell Biol. 28 (8), 2690-2700 (2008).</li><li>Sapountzi, V., Logan, I. R., Robson, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+functions+of+TIP60.">Cellular functions of TIP60.</a> Int J Biochem Cell Biol. 38 (9), 1496-1509 (2006).</li><li>Squatrito, M., Gorrini, C., Amati, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip60+in+DNA+damage+response+and+growth+control:+many+tricks+in+one+HAT.">Tip60 in DNA damage response and growth control: many tricks in one HAT.</a> Trends Cell Biol. 16 (9), 433-442 (2006).</li><li>Sun, Y., Xu, Y., Roy, K., Price, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage-induced+acetylation+of+lysine+3016+of+ATM+activates+ATM+kinase+activity.">DNA damage-induced acetylation of lysine 3016 of ATM activates ATM kinase activity.</a> Mol Cell Biol. 27 (24), 8502-8509 (2007).</li><li>Sykes, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+of+the+p53+DNA-binding+domain+regulates+apoptosis+induction.">Acetylation of the p53 DNA-binding domain regulates apoptosis induction.</a> Mol Cell. 24 (6), 841-851 (2006).</li><li>Tang, Y., Luo, J., Zhang, W., Gu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip60-dependent+acetylation+of+p53+modulates+the+decision+between+cell-cycle+arrest+and+apoptosis.">Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis.</a> Mol Cell. 24 (6), 827-839 (2006).</li><li>Chen, G., Cheng, Y., Tang, Y., Martinka, M., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Tip60+in+human+melanoma+cell+migration,+metastasis,+and+patient+survival.">Role of Tip60 in human melanoma cell migration, metastasis, and patient survival.</a> J Invest Dermatol. 132 (11), 2632-2641 (2012).</li><li>Gorrini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip60+is+a+haplo-insufficient+tumour+suppressor+required+for+an+oncogene-induced+DNA+damage+response.">Tip60 is a haplo-insufficient tumour suppressor required for an oncogene-induced DNA damage response.</a> Nature. 448 (7157), 1063-1067 (2007).</li><li>Jha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Destabilization+of+TIP60+by+human+papillomavirus+E6+results+in+attenuation+of+TIP60-dependent+transcriptional+regulation+and+apoptotic+pathway.">Destabilization of TIP60 by human papillomavirus E6 results in attenuation of TIP60-dependent transcriptional regulation and apoptotic pathway.</a> Mol Cell. 38 (5), 700-711 (2010).</li><li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+a+metastasis+suppressor+gene+by+Tip60+and+beta-catenin+complexes.">Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes.</a> Nature. 434 (7035), 921-926 (2005).</li><li>Sakuraba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+Tip60+gene+as+a+potential+marker+for+the+malignancy+of+colorectal+cancer.">Down-regulation of Tip60 gene as a potential marker for the malignancy of colorectal cancer.</a> Anticancer Res. 29 (10), 3953-3955 (2009).</li><li>Subbaiah, V. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E3+ligase+EDD1/UBR5+is+utilized+by+the+HPV+E6+oncogene+to+destabilize+tumor+suppressor+TIP60.">E3 ligase EDD1/UBR5 is utilized by the HPV E6 oncogene to destabilize tumor suppressor TIP60.</a> Oncogene. , (2015).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Nguyen, D. X., Bos, P. D., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nat Rev Cancer. 9 (4), 274-284 (2009).</li><li>Zijl, F., Krupitza, G., Mikulits, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+steps+of+metastasis:+cell+invasion+and+endothelial+transmigration.">Initial steps of metastasis: cell invasion and endothelial transmigration.</a> Mutat Res. 728 (1-2), 23-34 (2011).</li><li>Lamouille, S., Xu, J., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+epithelial-mesenchymal+transition.">Molecular mechanisms of epithelial-mesenchymal transition.</a> Nat Rev Mol Cell Biol. 15 (3), 178-196 (2014).</li><li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat Protoc. 2 (2), 329-333 (2007).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIP60+inhibits+metastasis+by+ablating+DNMT1-SNAIL2-driven+epithelial-mesenchymal+transition+program.">TIP60 inhibits metastasis by ablating DNMT1-SNAIL2-driven epithelial-mesenchymal transition program.</a> J Mol Cell Biol. , (2016).</li><li>Riahi, R., Yang, Y., Zhang, D. D., Wong, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+wound-healing+assays+for+probing+collective+cell+migration.">Advances in wound-healing assays for probing collective cell migration.</a> J Lab Autom. 17 (1), 59-65 (2012).</li><li>Jonkman, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+the+wound+healing+assay+using+live-cell+microscopy.">An introduction to the wound healing assay using live-cell microscopy.</a> Cell Adh Migr. 8 (5), 440-451 (2014).</li></ol>

The authors declare that they have no competing financial interests.

It is known that in the process of cell migration, cells can either move in the form of adherent sheets; loose clusters; or single, isolated cells. The mode and dynamic of cell invasion can vary from one condition to another, and the phenotype can change within hours. The traditional wound-healing assay is based on snapshot pictures taken from a microscope with a long time interval, such as 12 or 24 h. This makes it difficult to capture the dynamic of wound closure and the pattern of cell movements.
<p class="jove_co...

HIV-Tat-interactive protein 60 kDa (TIP60) is a lysine acetyltransferase that can acetylate both histone and non-histone proteins1,2. Its functions are found to have implications in multiple signaling pathways, including transcription, DNA-damage repair, and apoptosis1,3,4,5,6,7,8. Furthermore, TIP60 is often downregulated in cancers, and its downregulation is correlated with cancer metastasis and poor survival rates9,10,11,12,13,14. Tumor metastasis is the major cause of cancer-related death. It is a multi-step process, and the initial step of metastasis involves the migration and invasion of tumor cells into adjacent tissues15,16. In order to do so, tumor cells first must detach from the primary tumor mass, either in the form of a collectively invading cell sheet or as detached single cells17. During this process, tumor cells often undergo epithelial to mesenchymal transition (EMT), resulting in changes in morphology and cell adhesion capability17,18.
A few techniques have been developed to study the migration and invasion ability of tumor cells in vitro. Among them, the wound-healing assay is the most efficient and economical19. First, this method involves the creation of an artificial gap on a confluent monolayer of cells, thus allowing cells to migrate and close the gap. Second, images of the gaps can be captured at the beginning and end of the experiment. Finally, a comparison of gap closure is used to determine the rate of cell migration. A phase-contrast microscope is usually used to capture images for conventional wound-healing assays. Another advantage of the wound-healing assay is that it partly resembles in vivo tumor cell migration. Similarly to in vivo tumor metastasis, cell migration in wound-healing assays shows both the collective migration of epithelial sheets and the migration of detached single cells.
However, cell migration is known to be dynamic, and there is a need to document the movement of cells in real time. For instance, a conventional wound-healing assay does not allow a researcher to analyze single-cell movement. On the other hand, cells cultured for wound-healing assays are often serum-starved to inhibit cell proliferation. This is done to rule out the possibility of gap closure due to cell proliferation. Nonetheless, there will still be some proliferation and cell death, and phase-contrast images taken before and after the experiment are unable to differentiate between them.
The live-cell imaging technique addresses this limitation of conventional wound-healing assays. Using a live-imaging microscope combined with a CO2 incubator and appropriate temperature control, researchers can measure the gap size and its rate of closure over time while tracing the movement of actively migrating cells located at the tips of the invasive front. Hence, the implementation of live-cell imaging to monitor cell migration will not only provide better visualization of cell migration, but will also allow for the possibility to differentiate between cell migration and cell proliferation, thus providing a more reliable analysis of cell migration.
In this study, MCF10A breast epithelial cells were used to demonstrate the combination of the wound-healing assay and live-cell imaging to study the role of TIP60 in cell migration. The depletion of TIP60 results in increased migration abilities in MCF10A cells.

<table><tbody><tr><td>MCF10A cell</td><td>ATCC</td><td>CRL-10317TM</td><td>Breast epithelial cell</td></tr><tr><td>DMEM/F12 media (1:1)</td><td>Gibco</td><td>11330-032</td><td>MCF10A culture media</td></tr><tr><td>Epithelial growth factor (EGF)</td><td>Peprotech</td><td>AF-100-15</td><td>Supplements for MCF10A culture media</td></tr><tr><td>Cholera Toxin</td><td>Sigma-Aldrich</td><td>C-8052</td><td>Supplements for MCF10A culture media</td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H-0888</td><td>Supplements for MCF10A culture media</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I-1882</td><td>Supplements for MCF10A culture media</td></tr><tr><td>House serum</td><td>Gibco</td><td>16050-122</td><td>Supplements for MCF10A culture media</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td>For MCF10A detach from plate</td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td>267110</td><td>For counting cell</td></tr><tr><td>Opti-MEM reduced serum media</td><td>Gibco</td><td>31985-070</td><td>For siRNA mixture</td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen/Life Technologies</td><td>56532</td><td>Transfect reagent</td></tr><tr><td>Trizol reagent</td><td>Invitrogen/Life Technologies</td><td>15596-026</td><td>For mRNA extraction</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad</td><td>170-8891</td><td>For cDNA generation</td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Bio-Rad</td><td>172-5125</td><td>For real time qPCR</td></tr><tr><td>7500 Fast Real Time PCR system</td><td>Biosystems</td><td></td><td>Real time qPCR mechine</td></tr><tr><td>10-cm dish</td><td>Greiner bio-one</td><td>664160</td><td>For Knockdown experiment</td></tr><tr><td>24-well plate</td><td>Cellstar</td><td>662160</td><td>For wound healing assay</td></tr><tr><td>siControl siRNA</td><td>Self designed</td><td>Self designed</td><td>CGUACGCGGAAUACUUCGAdTdT</td></tr><tr><td>siTIP60 siRNA</td><td>Self designed</td><td>Self designed</td><td>UGAUCGAGUUCAGCUAUGAdTdT</td></tr><tr><td>Live cell observer</td><td>Zeiss</td><td></td><td>Zeiss inverted Cell Observer for live cell experiments</td></tr></tbody></table>

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.

General Schema of Live-cell Imaging-based Cell Migration Assay 
Figure 1A is a schema of this protocol. MCF10A cells transfected with siControl showed typical epithelial morphology. After the depletion of TIP60, MCF10A cells were more mesenchymal compared to control cells (Figure 1B).
Examination of TIP60 Knockdown Efficiency ...

Watch this Scientific Journal Video about Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60 at JoVE.com

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

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 ...

live-imaging-breast-epithelial-cell-migration-after-transient

Research

JoVE Journal

Cancer Research

6.8K Views.  National University of Singapore. 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. 

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

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Developmental Biology

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-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Bioengineering

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicine

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

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

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

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

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

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

Summary

Explore More Videos

Chapters in this video

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

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

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

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