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...

<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>

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 border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">MCF10A cell</td>
			<td style="width:211px;">ATCC</td>
			<td style="width:119px;">CRL-10317TM</td>
			<td style="width:257px;">Breast epithelial cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DMEM/F12 media (1:1)</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">11330-032</td>
			<td>MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Epithelial growth factor (EGF)</td>
			<td style="width:211px;">Peprotech</td>
			<td style="width:119px;">AF-100-15</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cholera Toxin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">C-8052</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hydrocortisone</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">H-0888</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Insulin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">I-1882</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">House serum</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">16050-122</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Trypsin</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>For MCF10A detach from plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hemacytometer</td>
			<td style="width:211px;">Fisher Scientific</td>
			<td style="width:119px;">267110</td>
			<td>For counting cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Opti-MEM reduced serum media</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>For siRNA mixture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Lipofectamine RNAiMAX</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">56532</td>
			<td>Transfect reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Trizol reagent</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">15596-026</td>
			<td>For mRNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">iScript cDNA Synthesis Kit</td>
			<td style="width:211px;">Bio-Rad</td>
			<td style="width:119px;">170-8891</td>
			<td>For cDNA generation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">iTaq Universal SYBR Green Supermix</td>
			<td style="width:211px;">Bio-Rad</td>
			<td style="width:119px;">172-5125</td>
			<td>For real time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">7500 Fast Real Time PCR system</td>
			<td style="width:211px;">Biosystems</td>
			<td style="width:119px;"></td>
			<td>Real time qPCR mechine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">10-cm dish</td>
			<td style="width:211px;">Greiner bio-one</td>
			<td style="width:119px;">664160</td>
			<td>For Knockdown experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">24-well plate</td>
			<td style="width:211px;">Cellstar</td>
			<td style="width:119px;">662160</td>
			<td>For wound healing assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siControl siRNA</td>
			<td style="width:211px;">Self designed</td>
			<td style="width:119px;">Self designed</td>
			<td>CGUACGCGGAAUACUUCGAdTdT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siTIP60 siRNA</td>
			<td style="width:211px;">Self designed</td>
			<td style="width:119px;">Self designed</td>
			<td>UGAUCGAGUUCAGCUAUGAdTdT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Live cell observer</td>
			<td style="width:211px;">Zeiss</td>
			<td style="width:119px;"></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 ...

The overall goal of this procedure is to observe the effects of the depletion of specific genes of interest on cell migration. This method can help us answer key questions in cancer metastasis, such as cancer cell migration. The main advantage of this technique is that it allows the dynamic cell migration to be monitored in real time.We first had the idea for this method, when we were observing cell morphology changes upon the patient of TIP60, a tumor suppressor. Begin by seeding one million breast epithelial cells in three milliliters of culture medium per 10 centimeter dish. Add 20 minutes room temperature incubated SRINA mixture drop by drop to the experimental cell culture.Manually shake both dishes to ensure that the cells are evenly distributed before placing them into a 37 degree Celsius incubator. After six hours, replace the supernatants with eight milliliters of fresh culture medium per dish and return the cultures to the incubator. The next morning, replace the supernatants with three milliliters of fresh culture medium per dish and add a second patch of SIRNA mixture to the experimental cell culture, as just demonstrated, shaking the dish to ensure the reagents are fully mixed before returning the cells to the incubator.Then replace the supernatants with eight milliliters of fresh culture medium after six hours and incubate the cultures over night. On day three, wash the cells one time with two milliliters of BPS per plate and detach the cells in both plates with one milliliter of tripson EDTA per dish for 20 minutes at 37 degrees Celsius. At the end of the incubation, stop the reaction with four milliliters of complete culture medium per plate.Resuspend the cells by pipetting and transfer the cell suspensions into individual 15 milliliter tubes. Collect the cells by centrifugation and resuspend the pellets in five milliliters of serum-free medium per tube. After counting, dilute both cell cultures to a 600, 000 cells per milliliter concentration and add 500 microliters of cells from each culture to one well of a 24 well plate to ensure 100%confluency.When all of the cells have been seeded, gently shake the plate back and forth and side to side to ensure even distribution and place the plate in the cell culture incubator for 24 hours. To check the knock down efficiency of the gene of interest, collect the remaining cells in the collection tubes with another centrifugation and isolate the RNA from the pellets, using a commercial reagent according to the manufacturers instructions for real time quantitative PCR evaluation. The next morning, at least one hour before beginning the live cell imaging, turn on the microscope and temperature control.Set the temperature to 37 degrees Celsius and carbon dioxide gas supply to 5%When the chamber has reached 37 degrees Celsius, use phase contrast microscopy to confirm that the cells are confluent and fully attached to the bottom of the wells. Then use a 200 microliter pipette tip to gently scratch a straight line across the entire length of the center of each well and gently wash all of the wells five times with serum free medium to remove the detached cells. After the last wash, add one milliliter of serum free medium to each well.Place the plate on the microscope stage under the 10 power objective and direct the emitted light to the eye pieces instead of the camera. Manually adjust the focus to locate each wound and the cells around it. Then return the emitted light back to the camera and use a live cell observing system to mark the position of the wounds and to set the time conditions.Image the cells for the appropriate experiment period, then open the first avi video in the appropriate imaging software and open the m track j plug-in. Click on the add tab to add a track for the single cells and choose one cell at the tip of the invasive front and one cell that can migrate on its own. Follow the movement of the chosen cells on the video using mtrackJ to trace their trafficking.Then click on the measure tab to measure the distance of the cell movement and save the videos with the tracks in avi format. After depletion of TIP60, breast epithelial cells are more mesenchymal compared to control cultured cells. Indeed, TIP60 expression, as well as epithelial cell adhesion molecule expression, decreases significantly after siTIP60 treatment.On the other hand, the expression of specific epithelial to mesenchymal transition driver genes is increased upon TIP60 depletion. Live imaging of the cell cultures after wound administration demonstrates a faster cell migration of siTIP60 treated cells, compared to siControl cultures, with a significant increase in the percentage of migrating cells observed in response to TIP60 depletion. Further, siControl cells move as a sheet, whereas more single cell movement is observed within the siTIP60 cultures.After watching this video, you should have a good understanding of how to use a live cell imaging technique to monitor dynamic changes of cell migration.

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

Research

JoVE Journal

Cancer Research

6.8K visualizações.  National University of Singapore. The overall goal of this procedure is to observe the effects of the depletion of specific genes of interest on cell migration. This method can help us answer key questions in cancer metastasis, such as cancer cell migration. The main advantage of this technique is that it allows the dynamic cell migration to be monitored in real time.We first had the idea for this method, when we were observing cell morphology changes upon the patient of TIP60, a tumor suppressor. Begin by seeding one ...