Name: xenopus laevis egg extract preparation and live imaging methods for visualizing dynamic cytoplasmic organization
Uploaded: 2021-06-06T13:00:00
Description: Watch this Scientific Journal Video about Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization at JoVE.com

We thank J. Kamenz, Y. Chen, and W. Y. C. Huang for comments on the manuscript. This work was supported by grants from the National Institutes of Health (R01 GM110564, P50 GM107615, and R35 GM131792) awarded to James E. Ferrell, Jr.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University.
1. Preparation of slides and coverslips
<ol>
	<li>Apply a layer of fluorinated ethylene propylene (FEP) adhesive tape to a glass slide with a roller applicator. Cut off excessive tape over the edges with a clean razor blade. Prepare FEP tape-coated coverslips in the same way (Figure 1A).</li>
	<li>Apply a double-sided sticky imaging sp...

<ol>
	<li>Murray, A. W., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+synthesis+drives+the+early+embryonic+cell+cycle.">Cyclin synthesis drives the early embryonic cell cycle.</a> Nature. 339 (6222), 275-280 (1989).</li><li>Dunphy, W. G., Brizuela, L., Beach, D., Newport, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus+cdc2+protein+is+a+component+of+MPF,+a+cytoplasmic+regulator+of+mitosis.">The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis.</a> Cell. 54 (3), 423-431 (1988).</li><li>Minshull, J., Golsteyn, R., Hill, C. S., Hunt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+A-+and+B-type+cyclin+associated+cdc2+kinases+in+Xenopus+turn+on+and+off+at+different+times+in+the+cell+cycle.">The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle.</a> EMBO J. 9 (9), 2865-2875 (1990).</li><li>Wu, J. Q., 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=PP1-mediated+dephosphorylation+of+phosphoproteins+at+mitotic+exit+is+controlled+by+inhibitor-1+and+PP1+phosphorylation.">PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation.</a> Nature Cell Biology. 11 (5), 644-651 (2009).</li><li>Pomerening, J. R., Sontag, E. D., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+cell+cycle+oscillator:+hysteresis+and+bistability+in+the+activation+of+Cdc2.">Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2.</a> Nature Cell Biology. 5 (4), 346-351 (2003).</li><li>Mochida, S., Maslen, S. L., Skehel, M., Hunt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Greatwall+phosphorylates+an+inhibitor+of+protein+phosphatase+2A+that+is+essential+for+mitosis.">Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis.</a> Science. 330 (6011), 1670-1673 (2010).</li><li>Blow, J. J., Laskey, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+DNA+replication+in+nuclei+and+purified+DNA+by+a+cell-free+extract+of+Xenopus+eggs.">Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.</a> Cell. 47 (4), 577-587 (1986).</li><li>Lohka, M. J., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+in+vitro+of+sperm+pronuclei+and+mitotic+chromosomes+induced+by+amphibian+ooplasmic+components.">Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.</a> Science. 220 (4598), 719-721 (1983).</li><li>Cheng, X., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+emergence+of+cell-like+organization+in+Xenopus+egg+extracts.">Spontaneous emergence of cell-like organization in Xenopus egg extracts.</a> Science. 366 (6465), 631-637 (2019).</li><li>Nguyen, P. 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=Spatial+organization+of+cytokinesis+signaling+reconstituted+in+a+cell-free+system.">Spatial organization of cytokinesis signaling reconstituted in a cell-free system.</a> Science. 346 (6206), 244-247 (2014).</li><li>Good, M. C., Vahey, M. D., Skandarajah, A., Fletcher, D. A., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+volume+modulates+spindle+size+during+embryogenesis.">Cytoplasmic volume modulates spindle size during embryogenesis.</a> Science. 342 (6160), 856-860 (2013).</li><li>Hazel, 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=Changes+in+cytoplasmic+volume+are+sufficient+to+drive+spindle+scaling.">Changes in cytoplasmic volume are sufficient to drive spindle scaling.</a> Science. 342 (6160), 853-856 (2013).</li><li>Brownlee, C., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importin+alpha+Partitioning+to+the+Plasma+Membrane+Regulates+Intracellular+Scaling.">Importin alpha Partitioning to the Plasma Membrane Regulates Intracellular Scaling.</a> Cell. 176 (4), 805-815 (2019).</li><li>Nguyen, P. A., Field, C. M., 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=Prc1E+and+Kif4A+control+microtubule+organization+within+and+between+large+Xenopus+egg+asters.">Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters.</a> Molecular Biology of the Cell. 29 (3), 304-316 (2018).</li><li>Desai, A., Maddox, P. S., Mitchison, T. J., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphase+A+chromosome+movement+and+poleward+spindle+microtubule+flux+occur+At+similar+rates+in+Xenopus+extract+spindles.">Anaphase A chromosome movement and poleward spindle microtubule flux occur At similar rates in Xenopus extract spindles.</a> Journal of Cell Biology. 141 (3), 703-713 (1998).</li><li>Mitchison, T. 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=Roles+of+polymerization+dynamics,+opposed+motors,+and+a+tensile+element+in+governing+the+length+of+Xenopus+extract+meiotic+spindles.">Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles.</a> Molecular Biology of the Cell. 16 (6), 3064-3076 (2005).</li><li>Mitchison, T. 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=Bipolarization+and+poleward+flux+correlate+during+Xenopus+extract+spindle+assembly.">Bipolarization and poleward flux correlate during Xenopus extract spindle assembly.</a> Molecular Biology of the Cell. 15 (12), 5603-5615 (2004).</li><li>Murray, A. W., Desai, A. B., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+observation+of+anaphase+in+vitro.">Real time observation of anaphase in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (22), 12327-12332 (1996).</li><li>Deming, P., Kornbluth, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+apoptosis+in+vitro+using+the+Xenopus+egg+extract+reconstitution+system.">Study of apoptosis in vitro using the Xenopus egg extract reconstitution system.</a> Methods in Molecular Biology. 322, 379-393 (2006).</li><li>Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+extracts.">Cell cycle extracts.</a> Methods in Cell Biology. 36, 581-605 (1991).</li><li>Field, C. M., 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=Assembly+of+Spindles+and+Asters+in+Xenopus+Egg+Extracts.">Assembly of Spindles and Asters in Xenopus Egg Extracts.</a> Cold Spring Harbor Protocols. 2018 (6), (2018).</li><li>Guan, 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=A+robust+and+tunable+mitotic+oscillator+in+artificial+cells.">A robust and tunable mitotic oscillator in artificial cells.</a> Elife. 7, (2018).</li><li>Guan, Y., Wang, S., Jin, M., Xu, H., Yang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+Cell-cycle+Oscillations+in+Microemulsions+of+Cell-free+Xenopus+Egg+Extracts.">Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts.</a> Journal of Visualized Experiments. (139), e58240 (2018).</li><li>Oakey, J., Gatlin, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Encapsulation+of+Demembranated+Sperm+Nuclei+in+Xenopus+Egg+Extracts.">Microfluidic Encapsulation of Demembranated Sperm Nuclei in Xenopus Egg Extracts.</a> Cold Spring Harbor Protocols. 2018 (8), (2018).</li><li>Smythe, C., Newport, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+for+the+study+of+nuclear+assembly,+DNA+replication,+and+nuclear+breakdown+in+Xenopus+laevis+egg+extracts.">Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts.</a> Methods in Cell Biology. 35, 449-468 (1991).</li><li>Field, C. 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=Actin+behavior+in+bulk+cytoplasm+is+cell+cycle+regulated+in+early+vertebrate+embryos.">Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos.</a> Journal of Cell Science. 124, 2086-2095 (2011).</li><li>Chang, J. B., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+trigger+waves+and+the+spatial+coordination+of+the+Xenopus+cell+cycle.">Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle.</a> Nature. 500 (7464), 603-607 (2013).</li><li>Chang, J. B., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robustly+Cycling+Xenopus+laevis+Cell-Free+Extracts+in+Teflon+Chambers.">Robustly Cycling Xenopus laevis Cell-Free Extracts in Teflon Chambers.</a> Cold Spring Harbor Protocols. 2018 (8), (2018).</li><li>Chatterjee, S., Javier, M., Stochaj, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+nuclear+protein+traffic+in+mammalian+cells.">In vivo analysis of nuclear protein traffic in mammalian cells.</a> Experimental Cell Research. 236 (1), 346-350 (1997).</li><li>Mochida, S., Hunt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+is+required+to+release+Xenopus+egg+extracts+from+meiotic+M+phase.">Calcineurin is required to release Xenopus egg extracts from meiotic M phase.</a> Nature. 449 (7160), 336-340 (2007).</li><li>Heald, R., 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=Self-organization+of+microtubules+into+bipolar+spindles+around+artificial+chromosomes+in+Xenopus+egg+extracts.">Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.</a> Nature. 382 (6590), 420-425 (1996).</li><li>Helmke, K. J., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TPX2+levels+modulate+meiotic+spindle+size+and+architecture+in+Xenopus+egg+extracts.">TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts.</a> Journal of Cell Biology. 206 (3), 385-393 (2014).</li><li>Sawin, K. E., 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=Mitotic+spindle+assembly+by+two+different+pathways+in+vitro.">Mitotic spindle assembly by two different pathways in vitro.</a> Journal of Cell Biology. 112 (5), 925-940 (1991).</li><li>Belmont, L. D., Hyman, A. A., Sawin, K. E., 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=Real-time+visualization+of+cell+cycle-dependent+changes+in+microtubule+dynamics+in+cytoplasmic+extracts.">Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts.</a> Cell. 62 (3), 579-589 (1990).</li><li>Krauss, S. W., Lee, G., Chasis, J. A., Mohandas, N., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+protein+4.1+domains+essential+for+mitotic+spindle+and+aster+microtubule+dynamics+and+organization+in+vitro.">Two protein 4.1 domains essential for mitotic spindle and aster microtubule dynamics and organization in vitro.</a> Journal of Biological Chemistry. 279 (26), 27591-27598 (2004).</li><li>Wang, S., Romano, F. B., Field, C. M., Mitchison, T. J., Rapoport, T. A. <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+determine+ER+network+morphology+during+the+cell+cycle+in+Xenopus+egg+extracts.">Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts.</a> Journal of Cell Biology. 203 (5), 801-814 (2013).</li><li>Field, C. M., Nguyen, P. A., Ishihara, K., Groen, A. C., 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=Xenopus+egg+cytoplasm+with+intact+actin.">Xenopus egg cytoplasm with intact actin.</a> Methods in Enzymology. 540, 399-415 (2014).</li><li>Good, M. C., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Cellular+Extracts+from+Xenopus+Eggs+and+Embryos.">Preparation of Cellular Extracts from Xenopus Eggs and Embryos.</a> Cold Spring Harbor Protocols. 2018 (6), (2018).</li><li>Field, C. M., Pelletier, J. F., 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=Xenopus+extract+approaches+to+studying+microtubule+organization+and+signaling+in+cytokinesis.">Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis.</a> Methods in Cell Biology. 137, 395-435 (2017).</li></ol>

Xenopus laevis egg extracts have emerged as a powerful model system for imaging-based&#160;studies of various subcellular structures10,14,15,16,17,18,21,31,32,33,<sup ...

The cytoplasm constitutes the main volume of a cell and has a distinct organization. The ingredients of the eukaryotic cytoplasm can self-assemble into a wide range of spatial structures, such as microtubule asters and the Golgi apparatus, which in turn are dynamically arranged and turned over depending on the cell&#39;s identity and physiological state. Understanding the spatial organization of the cytoplasm and its link to cellular functions is thus important for understanding how the cell works. Xenopus laevis egg extracts have traditionally been used for bulk biochemical assays1,2,3,4,5,6,7,8, but recent work establishes them as a powerful live imaging system for mechanistic studies of cytoplasmic structures and their cellular functions9,10,11,12,13,14,15,16,17,18. These undiluted extracts preserve many structures and functions of the cytoplasm, while allowing direct manipulations of cytoplasmic contents not achievable in conventional cell-based models19,20. This makes them ideal for characterizing cytoplasmic phenomena and dissecting their mechanistic underpinnings.
Existing methods for imaging extracts require chemical surface modifications, or fabrication of microfluidic devices. One coverslip-based method requires polyethylene glycol (PEG) passivation of glass coverslips21. A microemulsion-based method requires vapor deposition of trichloro(1H,1H,2H,2H-perfluorooctyl)silane on glass surfaces22,23. Microfluidic-based systems allow precise control of the volume, geometry and composition of extract droplets, but require specialized microfabrication facilities11,12,24.
Here we introduce an alternative method for imaging egg extracts that is easy to implement and utilizes readily available, low-cost materials. This includes preparation of an imaging chamber with a slide and a coverslip coated with fluorinated ethylene propylene (FEP) tape. The chamber can be used for imaging extracts with a variety of microscopy systems, including stereoscopes and upright and inverted microscopes. This method requires no chemical treatment of surfaces while achieving similar optical clarity obtained with existing glass-based methods discussed above. It is designed to image a layer of extracts with a uniform thickness across a 2D field, and can be easily extended to image a 3D volume of extracts. It is well suited for time-lapse imaging of collective cytoplasmic behavior over a large field of view.
We have used interphase-arrested egg extracts to demonstrate our imaging method. The extract preparation follows the protocol of Deming and Kornbluth19. Briefly, eggs naturally arrested in metaphase of meiosis II are crushed by a low speed spin. This spin releases the cytoplasm from meiotic arrest and allows the extract to proceed into interphase. Normally, cytochalasin B is added prior to the crushing spin to inhibit F-actin formation. However, it can be omitted if F-actin is desired. Cycloheximide is also added prior to the crushing spin to prevent the interphase extract from entering the next mitosis. The extracts are subsequently placed in the aforementioned imaging chambers and placed on a microscope. Finally, images are recorded over time at defined intervals by a camera connected to the microscope, producing time-lapse image series that capture the dynamical behavior of the extract in a 2D field.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>17 ml centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>337986</td>
			<td></td>
		</tr>
		<tr>
			<td>22x22 mm square #1 cover glass</td>
			<td>Corning</td>
			<td>284522</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>MilliporeSigma</td>
			<td>10236624001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>MilliporeSigma</td>
			<td>01810</td>
			<td>Protein synthesis inhibitor</td>
		</tr>
		<tr>
			<td>Cytochalasin B</td>
			<td>MilliporeSigma</td>
			<td>C6762</td>
			<td>Actin polymerization inhibitor</td>
		</tr>
		<tr>
			<td>Female Xenopus laevis frogs</td>
			<td>Nasco</td>
			<td>LM00535MX</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent HiLyte 488 labeled tubulin protein</td>
			<td>Cytoskeleton, Inc.</td>
			<td>TL488M-A</td>
			<td>For visualizing the microtubule cytoskeleton</td>
		</tr>
		<tr>
			<td>Fluorescent HiLyte 647 labeled tubulin protein</td>
			<td>Cytoskeleton, Inc.</td>
			<td>TL670M-A</td>
			<td>For visualizing the microtubule cytoskeleton</td>
		</tr>
		<tr>
			<td>Fluorinated ethylene propylene (FEP) optically clear tape</td>
			<td>CS Hyde company</td>
			<td>23-FEP-2-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin (hCG)</td>
			<td>MilliporeSigma</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging spacer</td>
			<td>Electron Microscopy Sciences</td>
			<td>70327-8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>MilliporeSigma</td>
			<td>11017101001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-518-100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>MilliporeSigma</td>
			<td>330760</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red CMXRos</td>
			<td>Thermo Fisher Scientific</td>
			<td>M7512</td>
			<td>For visualizing mitochondria</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin (PMSG)</td>
			<td>BioVendor</td>
			<td>RP1782725000</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller applicator</td>
			<td>Amazon</td>
			<td>B07HMBJSP8</td>
			<td>For applying the FEP tape to the glass slides and coverslips</td>
		</tr>
		<tr>
			<td>Single-edged razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>For removing excessive FEP tape</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
	</tbody>
</table>

xenopus laevis egg extract preparation and live imaging methods for visualizing dynamic cytoplasmic organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis egg extracts can be used to study the self-organization of the cytoplasm during interphase. Figure 2A shows results from a successful experiment. We supplemented interphase-arrested extracts with demembranated Xenopus laevis sperm nuclei19 at a concentration of 27 nuclei/&#181;L and 0.38 &#181;M purified GST-GFP-NLS27,28,<sup class="x...

Watch this Scientific Journal Video about Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization at JoVE.com

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic ...

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Biology

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Reconstitution Of ÃƒÅ½Ã‚Â²-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...