This research is supported by funding from CBCF (to ZW).

1. Preparing the Cells for Live Imaging
<ol>
	<li>Cell culture and seeding
	<ol>
		<li>Use MCF-7 breast cancer cells selected for resistance to docetaxel (MCF-7TXT) and their non-resistant parental cell line (MCF-7CC). The detailed selection process and the characterization of these selected cell lines were described previously24.</li>
		<li>Grow all cells in 10 cm culture dishes at 37 &#176;C in a medium composed 90% of Dulbecco&#39;s modified Eagle&#39;s ...

<ol>
	<li>Kamangar, F., Dores, G. M., Anderson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+cancer+incidence,+mortality,+and+prevalence+across+five+continents:+defining+priorities+to+reduce+cancer+disparities+in+different+geographic+regions+of+the+world.">Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world.</a> J Clin Oncol. 24 (14), 2137-2150 (2006).</li><li>Yardley, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+resistance+and+the+role+of+combination+chemotherapy+in+improving+patient+outcomes.">Drug resistance and the role of combination chemotherapy in improving patient outcomes.</a> Int J Breast Cancer. 2013, 137414 (2013).</li><li>Jassem, J., 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=Doxorubicin+and+paclitaxel+versus+fluorouracil,+doxorubicin,+and+cyclophosphamide+as+first-line+therapy+for+women+with+metastatic+breast+cancer:+final+results+of+a+randomized+phase+III+multicenter+trial.">Doxorubicin and paclitaxel versus fluorouracil, doxorubicin, and cyclophosphamide as first-line therapy for women with metastatic breast cancer: final results of a randomized phase III multicenter trial.</a> J Clin Oncol. 19 (6), 1707-1715 (2001).</li><li>Nabholtz, J. 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=Docetaxel+and+doxorubicin+compared+with+doxorubicin+and+cyclophosphamide+as+first-line+chemotherapy+for+metastatic+breast+cancer:+results+of+a+randomized,+multicenter,+phase+III+trial.">Docetaxel and doxorubicin compared with doxorubicin and cyclophosphamide as first-line chemotherapy for metastatic breast cancer: results of a randomized, multicenter, phase III trial.</a> J Clin Oncol. 21 (6), 968-975 (2003).</li><li>Zelnak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+taxane+and+anthracycline+resistance.">Overcoming taxane and anthracycline resistance.</a> Breast J. 16 (3), 309-312 (2010).</li><li>Rivera, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+anthracycline-resistant+and+taxane-resistant+metastatic+breast+cancer+and+new+therapeutic+options.">Implications of anthracycline-resistant and taxane-resistant metastatic breast cancer and new therapeutic options.</a> Breast J. 16 (3), 252-263 (2010).</li><li>Longley, D. B., Johnston, P. G. <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+drug+resistance.">Molecular mechanisms of drug resistance.</a> J Pathol. 205 (2), 275-292 (2005).</li><li>Downing, K. H., Nogales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallographic+structure+of+tubulin:+implications+for+dynamics+and+drug+binding.">Crystallographic structure of tubulin: implications for dynamics and drug binding.</a> Cell Struct.Funct. 24 (5), 269-275 (1999).</li><li>McGrogan, B. T., Gilmartin, B., Carney, D. N., McCann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxanes,+microtubules+and+chemoresistant+breast+cancer.">Taxanes, microtubules and chemoresistant breast cancer.</a> Biochim.Biophys.Acta. 1785 (2), 96-132 (2008).</li><li>Kamath, K., Oroudjev, E., Jordan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+microtubule+dynamic+instability+in+living+cells.">Determination of microtubule dynamic instability in living cells.</a> Methods Cell Biol. 97, 1-14 (2010).</li><li>Dumontet, C., Jordan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-binding+agents:+a+dynamic+field+of+cancer+therapeutics.">Microtubule-binding agents: a dynamic field of cancer therapeutics.</a> Nat Rev Drug Discov. 9 (10), 790-803 (2010).</li><li>Jordan, M. A., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+as+a+target+for+anticancer+drugs.">Microtubules as a target for anticancer drugs.</a> Nat.Rev.Cancer. 4 (4), 253-265 (2004).</li><li>Gascoigne, K. E., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+anti-mitotic+drugs+kill+cancer+cells?.">How do anti-mitotic drugs kill cancer cells?.</a> J.Cell Sci. 122 (15), 2579-2585 (2009).</li><li>Kavallaris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+and+resistance+to+tubulin-binding+agents.">Microtubules and resistance to tubulin-binding agents.</a> Nat.Rev.Cancer. 10 (3), 194-204 (2010).</li><li>Diaz, J. F., Valpuesta, J. M., Chacon, P., Diakun, G., Andreu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+microtubule+protofilament+number+induced+by+Taxol+binding+to+an+easily+accessible+site.+Internal+microtubule+dynamics.">Changes in microtubule protofilament number induced by Taxol binding to an easily accessible site. Internal microtubule dynamics.</a> J.Biol.Chem. 273 (50), 33803-33810 (1998).</li><li>Abal, M., Andreu, J. M., Barasoain, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxanes:+microtubule+and+centrosome+targets,+and+cell+cycle+dependent+mechanisms+of+action.">Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action.</a> Curr Cancer Drug Targets. 3 (3), 193-203 (2003).</li><li>Wang, 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=Multiple+mechanisms+underlying+acquired+resistance+to+taxanes+in+selected+docetaxel-resistant+MCF-7+breast+cancer+cells.">Multiple mechanisms underlying acquired resistance to taxanes in selected docetaxel-resistant MCF-7 breast cancer cells.</a> BMC Cancer. 14 (37), (2014).</li><li>Lal, S., Mahajan, A., Chen, W. N., Chowbay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacogenetics+of+target+genes+across+doxorubicin+disposition+pathway:+a+review.">Pharmacogenetics of target genes across doxorubicin disposition pathway: a review.</a> Curr. Drug Metab. 11 (1), 115-128 (2010).</li><li>Murray, S., Briasoulis, E., Linardou, H., Bafaloukos, D., Papadimitriou, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxane+resistance+in+breast+cancer:+mechanisms,+predictive+biomarkers+and+circumvention+strategies.">Taxane resistance in breast cancer: mechanisms, predictive biomarkers and circumvention strategies.</a> Cancer Treat.Rev. 38 (7), 890-903 (2012).</li><li>Kamath, K., Wilson, L., Cabral, F., Jordan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BetaIII-tubulin+induces+paclitaxel+resistance+in+association+with+reduced+effects+on+microtubule+dynamic+instability.">BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability.</a> J.Biol.Chem. 280 (13), 12902-12907 (2005).</li><li>Banerjee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+levels+of+tyrosinated+alpha-,+beta(III)-,+and+beta(IV)-tubulin+isotypes+in+paclitaxel-resistant+MCF-7+breast+cancer+cells.">Increased levels of tyrosinated alpha-, beta(III)-, and beta(IV)-tubulin isotypes in paclitaxel-resistant MCF-7 breast cancer cells.</a> Biochem.Biophys.Res.Commun. 293 (1), 598-601 (2002).</li><li>Wiesen, K. M., Xia, S., Yang, C. P., Horwitz, 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=Wild-type+class+I+beta-tubulin+sensitizes+Taxol-resistant+breast+adenocarcinoma+cells+harboring+a+beta-tubulin+mutation.">Wild-type class I beta-tubulin sensitizes Taxol-resistant breast adenocarcinoma cells harboring a beta-tubulin mutation.</a> Cancer Lett. 257 (2), 227-235 (2007).</li><li>Iseri, O. D., Kars, M. D., Arpaci, F., Gunduz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+of+drug-resistant+MCF-7+cells:+implications+for+relation+to+extracellular+matrix+proteins.">Gene expression analysis of drug-resistant MCF-7 cells: implications for relation to extracellular matrix proteins.</a> Cancer Chemother.Pharmacol. 65 (3), 447-455 (2010).</li><li>Hembruff, S. L., 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=Role+of+drug+transporters+and+drug+accumulation+in+the+temporal+acquisition+of+drug+resistance.">Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance.</a> BMC.Cancer. 8, 318 (2008).</li><li>Yenjerla, M., Lopus, M., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+dynamic+instability+of+steady-state+microtubules+in+vitro+by+video-enhanced+differential+interference+contrast+microscopy+with+an+appendix+by+Emin+Oroudjev.">Analysis of dynamic instability of steady-state microtubules in vitro by video-enhanced differential interference contrast microscopy with an appendix by Emin Oroudjev.</a> Methods Cell Biol. 95, 189-206 (2010).</li><li>Sammak, P. J., Gorbsky, G. J., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+in+vivo:+a+test+of+mechanisms+of+turnover.">Microtubule dynamics in vivo: a test of mechanisms of turnover.</a> J Cell Biol. 104 (3), 395-405 (1987).</li><li>Walker, R. A., 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=Dynamic+instability+of+individual+microtubules+analyzed+by+video+light+microscopy:+rate+constants+and+transition+frequencies.">Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.</a> J Cell Biol. 107 (4), 1437-1448 (1988).</li><li>Desai, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+polymerization+dynamics.">Microtubule polymerization dynamics.</a> Annu Rev Cell Dev Biol. 13, 83-117 (1997).</li><li>Walczak, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+and+tubulin+interacting+proteins.">Microtubule dynamics and tubulin interacting proteins.</a> Curr Opin Cell Biol. 12 (1), 52-56 (2000).</li></ol>

The authors declare that they have no competing financial interests.

There are two major methods to measure microtubule dynamic instability: in vitro and in vivo. In the in vitro method, purified tubulin is used to measure microtubule dynamic instability with computer-enhanced time-lapse differential interference-contrast microscopy. In the in vivo method, microinjected fluorescent tubulin, or expressed GFP-tubulin, is incorporated into microtubules. The dynamics (growth and shortening) of the microtubules is then recorded by time-lapse using a sensitiv...

The leading cause of breast cancer mortality is through metastasis1,2. Taxanes, such as docetaxel and paclitaxel, are currently used as first-line regimens in the treatment of metastatic breast cancer2,3,4,5,6. They are part of a group of microtubule-targeting agents (MTAs) that disrupt microtubule dynamics. However, one of the greatest challenges to using taxanes in curative therapy is the development of taxane resistance in cancer cells, which leads to disease recurrence7. Drug resistance accounts for more than 90% of all deaths among patients with metastatic breast cancer7.
Microtubules are formed by the polymerization of &#945;- and &#946;-tubulin heterodimers8,9. The precise regulation of microtubule dynamics is important for many cellular functions, including cell polarization, cell cycle progression, intracellular transport, and cell signaling. Dysregulation of microtubules and their dynamics will disrupt cell function and result in cell death10,11. Depending on how they cause this dysregulation, MTA drugs can be classified as either microtubule stabilizing agents (i.e., taxanes) or microtubule-destabalizing agents (i.e., vinca alkaloids or colchicine-site binding agents)20. Despite their opposite effects on microtubule mass, at a sufficient dosage, both classes can kill cancer cells through their effects on microtubule dynamics21.
Taxanes function primarily by stabilizing the microtubule spindle12, leading to chromosomal misalignment. The subsequent perpetual activation of the spindle assembly checkpoint (SAC) arrests the cell in mitosis. Prolonged mitotic arrest then causes apoptosis13,14. Taxane interacts with microtubules through the taxane binding site on &#946;-tubulin8,15, which is only present in assembled tubulin16.
Multiple mechanisms for taxane resistance have been proposed9,17. These mechanisms include both general multidrug resistance due to the overexpression of drug-efflux proteins and taxane-specific resistance5,9,18,19. For example, taxane-resistant cancer cells may have altered expression and function of certain &#946;-tubulin isotypes5,9,19,20,21,22,23. By using an in vivo method to measure microtubule dynamic instability, we show that, when compared to non-resistant, parental MCF-7CC cells17, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment.
To better understand the function of MTAs and the exact mechanism of taxane-resistance in cancerous cells, it is essential to measure microtubule dynamics. Here, we report an in vivo method of doing so. By using live imaging in combination with the expression of GFP-tagged tubulin in cells, we can measure the microtubule dynamics of MCF-7TXT and MCF-7CC cells with and without docetaxel treatment. The results can help us design more effective drugs that can overcome taxane resistance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Life Technologies, Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco, Invitrogen</td>
			<td>12483</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Anti (100x)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>docetaxel</td>
			<td>Sigma-Aldrich</td>
			<td>01885-5mg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM phenol red-free</td>
			<td>Gibco, Invitrogen</td>
			<td>21063</td>
			<td></td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td>
			<td>ThermoFisher Scientific</td>
			<td>C10613</td>
			<td>Key reagent for expressing GFP tubulin in cells</td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP</td>
			<td>ThermoFisher Scientific</td>
			<td>B10383</td>
			<td>Control</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich+B9:AA9</td>
			<td>472301</td>
			<td>for dissoving decetaxel</td>
		</tr>
		<tr>
			<td>22-mm glass coveslip</td>
			<td>Fisher Scientifics</td>
			<td>12-545-101</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>Greiner Bio-One International</td>
			<td>6 Well Celi Culture Plate</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Microscopy Imaging Systems</td>
			<td>GE Health</td>
			<td></td>
			<td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemacytometer Set, Hausser Scientific</td>
			<td>Hausser Scientific, Distributed by VWR</td>
			<td>Supplier No.: 1492 VWR No.:15170-172</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the protocol presented here, we studied the effects of docetaxel on the microtubule dynamics of normal (MCF-7CC) and docetaxel-resistant (MCF-7TXT) breast cancer cells. Two sets of images show the effects of docetaxel (0.5 &#181;M) on microtubule growth and shortening in MCF-7CC and MCF-7TXT cells (Figure 1A).
We also calculated the rate of microtubule growth or ...

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

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

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 ...

The overall goal of this experiment is to measure microtubule dynamic instability in docetaxel resistant breast cancer cells. This method can help answer key questions in the field of cell biology about microtubule dynamic instability under physiological conditions, and following various drug treatments. The main advantages of this technique are that it provides a simple, reliable and physiologically applicable protocol for studying microtubule dynamics in living cells.Before beginning the procedure, grow the breast cancer cells in complete culture medium in 10 centimeter culture dishes at 37 degrees Celsius, and 5%carbon dioxide to the appropriate degree of confluency. The day before the experiment, rinse 24 millimeter poly L-lysine coated glass cover slips with 70%alcohol and expose the slips to UV light overnight. The next morning, place one cover slip into each well of a six well plate.Then wash the cultures with PBS, and detach the cells in each dish with 0.5 milliliters of trypsin EDTA. When the cells have begun to lift from the plate bottom, neutralize the trypsin with five milliliters of fresh culture medium and count the cells. Then add approximately one times 10 the fifth cells to each cover slip, shake the plate gently, and return the cells to the incubator for at least 36 hours.To assay the microtubule dynamics of the cells mix GFP tagged alpha tubulin stock solution several times by inversion to ensure a homogenous solution without vortexing. Then replace the culture medium in each well with two milliliters of fresh medium containing 40 microliters of the tag tubulin. Shake the plate gently to allow complete mixing of the marker with the culture medium and return the plate to the incubator for another 24 hours.The next day, replace the culture medium with fresh medium containing docetaxel and return the plate to the incubator for 30 minutes. While the cells are being inhibited, turn on the inverted fluorescence microscope, and prewarm the sample holder to 37 degrees Celsius. Then select the 60X 1.42 NA oil objective lens and mount the first cover slip onto the sample holder.Cover the sample with one milliliter of fresh 37 degree Celsius docetaxel containing medium and locate a cluster of flat cells exhibiting a high GFP tagged alpha tubulin fluorescence intensity with clearly visible microtubule structures. Focus on the peripheral area of one of the cells in the cluster, and set the appropriate exposure time in the image acquisition frequency. Exactly 50 minutes after adding the first aliquot of docetaxel begin recording the microtubule dynamics for two minutes, obtaining a photo every two seconds.When all of the images have been acquired, image the next cell as just demonstrated, until images for five cells total from all three wells of each docetaxel concentration have been captured. When all of the images have been acquired for each inhibitor concentration open the deconvolution program, within the microscope system and click process. Next, select deconvolve and drag the image file to the input window.Click do to deconvolve the image, and open the deconvolved image file within the microscope software. Click file and save as movie. Set the movie format as AV and the animation style as forward.Set the compression quality to 100%and the frame rate to five per second. Then check the animate through time box and click do it to generate the video. In this representative experiment, normal and docetaxel resistant breast cancer cells were treated with docetaxel and imaged as just demonstrated, but the effects of the treatment on microtubule growth and shortening, most evident in the non docetaxel resistant cells.Indeed, measuring the rate of microtubule growth and shortening under 0.5 micromolar docetaxel treatment conditions reveals a strong inhibition of the tubulin dynamics of parental but not docetaxel resistant cells. Once mastered, the entire process can be completed within 60 hours if the techniques are performed properly. While attempting this procedure, it is important to remember to select flat cells, with a high GFP tubulin fluorescence intensity, and to reduce both the intensity of the excitation fluorescence and the duration of exposure to avoid fading of the GFP fluorescence.Since it's development, this technique has paved the way for researchers in the fields of cell and cancer biology to assess the effects of various microtubule targeting agents on normal and cancerous cells. After watching this video, you should have a good understanding of how to measure microtubule dynamic instability in living cells under a variety of conditions.

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7.4K vistas.  University of Alberta. The overall goal of this experiment is to measure microtubule dynamic instability in docetaxel resistant breast cancer cells. This method can help answer key questions in the field of cell biology about microtubule dynamic instability under physiological conditions, and following various drug treatments. The main advantages of this technique are that it provides a simple, reliable and physiologically applicable protocol for studying microtubule dynamics in living cells.Before beginning...