This study was supported by grants from the Japan Society for the Promotion of Science KAKENHI (https://www.jsps.go.jp/english/index.html) to N.U. (19K23758, 21K06295) and K.W. (19H03242, 20K21420, 21H00420), from the Ohsumi Frontier Science Foundation (https://www.ofsf.or.jp/en/) to K.W., and from the Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials (http://alliance.tagen.tohoku.ac.jp/english/) to N.U. and K.W. We thank Ms.&#160;Miyuki Shinohara (Hosei Univ.) for her help in preparing the figures.

A wild-type strain of Chlamydomonas reinhardtii, CC-125, was used for the present study. CC-125 was obtained from Chlamydomonas Resource Center (see Table of Materials) and maintained on a Tris-acetate-phosphate (TAP)16, 1.5% agarose medium at 20-25 &#176;C.
1. Cell culture
<ol>
	<li>Culture Chlamydomonas reinhardtii (CC-125) in TAP medium16 in a 12 h/12 h light-dark period (light conditions for the light period: ~50 &#181;mol photons m&#8722;2 s&#8722;1 white light) at 20-25 &#176;C for 2 days (Figure 1). 
	NOTE: The cells must be in a mid-logarithmic growth phase (Movie 1). Long culture (&#62;4 days, in the late-logarithmic growth or stationary phase) decreases the reactivation efficiency of demembranated cell models.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63869/63869fig01.jpg" /> 
Figure 1: Liquid culture after 2-day culturing. From a TAP-1.5% agar plate, a chunk of wild-type cells to fill the platinum loop was inoculated to ~150 mL TAP liquid medium in a flask. The cell density after 2-day culture was 2.3 &#215; 106 cells/mL. <a href="https://www.jove.com/files/ftp_upload/63869/63869fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 1: Swimming of live cells. Cells were observed under a microscope with a 10x objective lens and an oil-immersion dark-field condenser. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63869/newMovie1.mp4" target="_blank">Please click here to download this Movie.</a>
2. Preparation of demembranated cell models
NOTE: Before starting the experiment, keep the washing buffer at room temperature and the demembranation, dilution, reactivation buffers, and the ATP solution on ice. The composition of these buffers is provided in Supplementary Table 1.
<ol>
	<li>Centrifuge ~10 mL of liquid culture at 1000 &#215; g at 20 &#176;C for 3 min. 
	NOTE: Throughout the protocol from this step, do not use autoclaved plasticware (tubes, pipette tips, etc.), decreasing reactivation efficiency. For conical tubes, reused ones are preferable. The cell density after 2-day culture may be typically 1.0-5.0 &#215; 106 cells/mL. When the cell density is lower than this, take enough culture volume to contain ~5 &#215; 107 cells.</li>
	<li>Discard the supernatant, first by decantation and then by a Pasteur pipette, and resuspend the precipitates in ~5 mL of washing buffer.</li>
	<li>Centrifuge cells in the washing buffer at 1000 &#215; g for 3 min at 20 &#176;C.</li>
	<li>Discard the supernatant carefully with a pipette (Figure 2). 
	NOTE: Pasteur pipettes are preferable. When using micropipettes, avoid using pipette tips that have been autoclaved.</li>
	<li>Overlay ~0.5 mL of demembranation buffer on a cell pellet, shake the tube gently by hand to roughly suspend the cells in the buffer, and put the tube on ice (Figure 3A). 
	NOTE: It is not necessary to entirely suspend the pellet at this moment. Raise the concentration of MgSO4 to 15 mM in the demembranation and reactivation buffers when cell models are reactivated, with a final ATP concentration &#62;1 mM for stable reactivation17.</li>
	<li>Suspend the remaining cell pellet gently with a pipette, and place the tube on ice again (Figure 3B).</li>
	<li>Take 5-10 &#181;L of the cell models, dilute 10-fold with the dilution buffer, and observe under the microscope (Movie 2) to confirm all the cell models are demembranated and not swimming (Figure 4). 
	NOTE: If some cells are still swimming (alive), add the non-ionic detergent used in the demembranation buffer directly to the cell model solution to the final concentration of ~0.15%. Alternatively, repeat steps 2.1-2.5 with the demembranation solution containing 0.15% of detergent.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63869/63869fig02.jpg" /> 
Figure 2: Discarding the supernatant. The rest of the supernatant was carefully removed with a Pasteur pipette after the supernatant was removed by decantation of the tube. <a href="https://www.jove.com/files/ftp_upload/63869/63869fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63869/63869fig03.jpg" /> 
Figure 3: Demembranation. (A) After overlaying 0.5 mL of demembranation solution onto the cell pellet, the solution was mixed by a hand to suspend the cells roughly. (B) After mixing, the rest of the cell pellet was entirely suspended in the solution by a Pasteur pipette. <a href="https://www.jove.com/files/ftp_upload/63869/63869fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63869/63869fig04.jpg" /> 
Figure 4: Effects of demembranation. (A) A live cell stuck on a glass slide. (B) A cell model stuck on a glass slide. Note that in the cell model, the cilia became slightly thinner. The images were observed under a microscope with a 20x objective lens and an oil-immersion dark-field condenser. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63869/63869fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 2: Confirmation of demembranation. Cell model suspension was observed under a microscope with a 10x objective lens and an oil-immersion dark-field condenser. No cell was swimming. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63869/newMovie2.mp4" target="_blank">Please click here to download this Movie.</a>
3. Reactivation of demembranated cell models
<ol>
	<li>Mix 80 &#181;L of reactivation solution, 10 &#181;L of ATP solution, and 10 &#181;L of cell models in a 0.5 mL tube by tapping the tube (Figure 5). 
	NOTE: The final ATP concentration must be &#60;3 mM because ciliary beating frequency, a parameter for ciliary motion, increases with ATP concentration and saturates at 2-3 mM of ATP17. 
	CAUTION: Mixing by pipetting or vortexing causes deciliation and decreases reactivation efficiency.
	<ol>
		<li>For reactivating with &#60;0.2 mM of ATP, add an ATP-regeneration system, such as 70 U/mL of creatine kinase and 5 mM of creatine phosphate (see Table of Materials). 
		NOTE: The reactivation solution is prepared at a higher concentration (1.125x, Supplementary Table 1) than the dilution solution so that after mixing the ATP dissolved in water, the contents reach the following final concentrations: 30 mM of Hepes (pH 7.4), 5 mM of MgSO4, 1 mM of dithiothreitol (DTT), 1 mM of EGTA, and 50 mM of potassium acetate (see Table of Materials).</li>
	</ol>
	</li>
	<li>Put ~30 &#181;L of the mixed solution onto a glass slide and gently place a coverslip on it with spacers to avoid mechanical shock to the cell models (Figure 6). 
	NOTE: White petroleum or double-sided adhesive tapes can be used to make the spacers.</li>
	<li>Observe the reactivated cell models under a microscope (Movie 3).</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63869/63869fig05.jpg" /> 
Figure 5: Mixing solution by tapping the tube. (A) To 80 &#181;L of the reactivation solution, 10 &#181;L of ATP solution, and 10 &#181;L of cell models were added in sequential order. (B) The solution was mixed by tapping the tube with a finger. <a href="https://www.jove.com/files/ftp_upload/63869/63869fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63869/63869fig06.jpg" /> 
Figure 6: Making spacers on coverslip edges. (A) A thin layer of white petroleum was applied to the back of a hand. (B) A small amount of white petroleum was scraped off with an edge of a coverslip. (C) A spacer was made on the edge of a coverslip. (D) Another spacer was made on the opposite edge. <a href="https://www.jove.com/files/ftp_upload/63869/63869fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 3: Swimming of reactivated cell models. The motility of the cell models was reactivated by adding ATP at a final concentration of 1 mM, and observed under a microscope with a 10x objective lens and an oil-immersion dark-field condenser. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63869/newMovie3.mp4" target="_blank">Please click here to download this Movie.</a>

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S., Satir, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direction+of+active+sliding+of+microtubules+in+Tetrahymena+cilia.">Direction of active sliding of microtubules in Tetrahymena cilia.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (5), 2045-2049 (1977).</li><li>Fox, L. A., Sale, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direction+of+force+generated+by+the+inner+row+of+dynein+arms+on+flagellar+microtubules.">Direction of force generated by the inner row of dynein arms on flagellar microtubules.</a> Journal of Cell Biology. 105 (4), 1781-1787 (1987).</li><li>Kamiya, R., Yagi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversity+of+axonemal+dyneins+as+assessed+by+in+vitro+and+in+vivo+motility+assays+of+Chlamydomonas+mutants.">Functional diversity of axonemal dyneins as assessed by in vitro and in vivo motility assays of Chlamydomonas mutants.</a> Zoolog Science. 31 (10), 633-644 (2014).</li><li>Kamiya, R., Witman, G. 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S., 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=Isolated+flagellar+apparatus+of+Chlamydomonas:+characterization+of+forward+swimming+and+alteration+of+waveform+and+reversal+of+motion+by+calcium+ions+in+vitro.">Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of waveform and reversal of motion by calcium ions in vitro.</a> Journal of Cell Science. 33, 235-253 (1978).</li><li>Fujiu, K., Nakayama, Y., Yanagisawa, A., Sokabe, M., Yoshimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydomonas+CAV2+encodes+a+voltage-+dependent+calcium+channel+required+for+the+flagellar+waveform+conversion.">Chlamydomonas CAV2 encodes a voltage- dependent calcium channel required for the flagellar waveform conversion.</a> Current Biology. 19 (2), 133-139 (2009).</li><li>Bessen, M., Fay, R. B., Witman, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+control+of+waveform+in+isolated+flagellar+axonemes+of+Chlamydomonas.">Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas.</a> Journal of Cell Biology. 86 (2), 446-455 (1980).</li><li>Wakabayashi, K., King, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Chlamydomonas+reinhardtii+flagellar+motility+by+redox+poise.">Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise.</a> Journal of Cell Biology. 173 (5), 743-754 (2006).</li><li>Saegusa, Y., Yoshimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP+controls+the+balance+of+the+propulsive+forces+generated+by+the+two+flagella+of+Chlamydomonas.">cAMP controls the balance of the propulsive forces generated by the two flagella of Chlamydomonas.</a> Cytoskeleton. 72 (8), 412-421 (2015).</li><li>Gorman, D. S., Levine, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+f+and+plastocyanin:+their+sequence+in+the+photosynthetic+electron+transport+chain+of+Chlamydomonas+reinhardi.">Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi.</a> Proceedings of the National Academy of Sciences of the United States of America. 54 (6), 1665-1669 (1965).</li><li>Takano, W., Hisabori, T., Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+estimation+of+cytosolic+ATP+concentration+from+the+ciliary+beating+frequency+in+the+green+alga+Chlamydomonas+reinhardtii.">Rapid estimation of cytosolic ATP concentration from the ciliary beating frequency in the green alga Chlamydomonas reinhardtii.</a> Journal of Biological Chemistry. 296, 100156 (2021).</li><li>Wakabayashi, K., Yagi, T., Kamiya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-dependent+waveform+conversion+in+the+flagellar+axoneme+of+Chlamydomonas+mutants+lacking+the+central-pair/radial+spoke+system.">Ca2+-dependent waveform conversion in the flagellar axoneme of Chlamydomonas mutants lacking the central-pair/radial spoke system.</a> Cell Motility and the Cytoskeleton. 38 (1), 22-28 (1997).</li><li>Yueh, Y. 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The authors have nothing to disclose.

There are two critical steps in this protocol. The first is a process known as demembranation, which needs to be carried out gently but thoroughly. Deciliation (i.e., detaching of cilia from the cell body) is induced by vigorous pipetting or vortexing, rendering the cell models immobile even after the addition of ATP. Typically, 5 &#215; 107 cells are suspended in ~0.5 mL of demembranation buffer (final cell density: 1 &#215; 108 cells/mL). The reactivation rate of the cell models would decrease if ...

The in vitro reactivation of demembranated motile cells is a valuable tool for studying the molecular basis for the regulatory mechanism of cell motility. Szent-Gy&#246;rgyi first demonstrated in vitro contraction of rabbit skeletal muscle fibers extracted with 50% glycerol by adding adenosine triphosphate (ATP)1. This experiment was the first to prove that ATP energizes muscle contraction. The methodology was soon applied to the study of ATP-energized ciliary/flagellar motility, such as sperm flagella2, Paramecium cilia3, and Chlamydomonas reinhardtii cilia (also called flagella)4 using non-ionic detergents for demembranation.
The unicellular green alga C. reinhardtii is a model organism for studying cilia: it swims with two cilia by beating them like a human&#39;s breaststroke5. Ciliary motion is driven by dynein, a minus-end directed microtubule-based motor protein6,7. Ciliary dyneins can be classified into outer-arm dyneins and inner-arm dyneins. Mutants lacking each kind of dynein have been isolated as slow-swimming mutants with different motility abnormalities. Detailed in vitro motility analysis of these mutants has significantly advanced dynein research8.
Many important findings have been achieved utilizing this method and its derivatives since the in vitro reactivation experiment of demembranated C. reinhardtii cells (cell models) was established. Reactivation of cell models in a series of Ca2+ buffers, for example, showed9 that two cilia are differently regulated by submicromolar Ca2+, and this asymmetrical cilia control enables the phototactic orientation of C. reinhardtii10. Furthermore, both cilia show waveform conversion from the forward swimming mode (called asymmetric waveform) to the backward swimming mode (called symmetric waveform that appears for a short period when cells are photo- or mechano-shocked)11,12. This waveform conversion is regulated by submillimolar Ca2+, which was shown by the reactivation of the so-called nucleoflagellar apparatus (a complex containing two cilia, the basal bodies, the structures linking the basal bodies with the nucleus, and the remnant of the nucleus)11 or demembranated axonemes of isolated cilia13. Other than Ca2+, redox (reduction-oxidation) poise is a signal that regulates the ciliary beating frequency, which was shown by reactivation of cell models in redox buffers containing different ratios of reduced glutathione vs. oxidized glutathione14. In addition, cyclic adenosine monophosphate (cAMP) asymmetrically regulates two cilia, which was shown by the reactivation of axonemes with photocleavable caged cAMP15. These in vitro findings, combined with genetic findings, have led to a deeper understanding of the molecular mechanisms of cilia regulation in C. reinhardtii.
A protocol for reactivating the cell models is described here. The method is simple, allows for various modifications, and can be applied to multiple organisms that move with cilia. However, because demembranated cells are fragile, it requires some tips to reactivate the motility of cell models with good efficiency while preventing deciliation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mL plastic tube</td>
			<td>QSP</td>
			<td>502-PLN-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>SARSTEDT</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sima-Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>KUBOTA</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlamydomonas strain CC-125</td>
			<td>Chlamydomonas Resource Center</td>
			<td></td>
			<td>https://www.chlamycollection.org/</td>
		</tr>
		<tr>
			<td>Creatine kinase</td>
			<td>Merck</td>
			<td>CK-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine phosphate</td>
			<td>Merck</td>
			<td>CRPHO-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Nakalai tesque</td>
			<td>14128-46</td>
			<td></td>
		</tr>
		<tr>
			<td>GEDTA(EGTA)</td>
			<td>Dojindo</td>
			<td>G002</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Dojindo</td>
			<td>GB70</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td>IUPAC name is octylphenoxypolyethoxyethanol: IGEPAL CA-630 is a substitute for Nonidet P-40 (NP-40); NP-40 is no longer available in Sigma-Aldrich.</td>
		</tr>
		<tr>
			<td>MgSO4-7H2O</td>
			<td>Nakalai tesque</td>
			<td>21002-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>fisher scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol, Mr 20,000</td>
			<td>Merck</td>
			<td>8.18897.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium acetate</td>
			<td>Nakalai tesque</td>
			<td>28434-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Nacalai</td>
			<td>31511-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>196-00015</td>
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reactivation of demembranated cell models in chlamydomonas reinhardtii

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Gy&#246;rgyi demonstrated in the mid-20th century, in vitro reactivation of demembranated cells has been a traditional and potent way to examine cell motility. The fundamental advantage of this experimental method is that the composition of the reactivation solution may be easily changed. For example, a high-Ca2+ concentration environment that occurs only temporarily due to membrane excitation in vivo can be replicated in the lab. Eukaryotic cilia (a.k.a. flagella) are elaborate motility machinery whose regulatory mechanisms are still to be clarified. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism in the research field of cilia. The reactivation experiments using demembranated cell models of C. reinhardtii and their derivatives, such as demembranated axonemes of isolated cilia, have significantly contributed to understanding the molecular mechanisms of ciliary motility. Those experiments clarified that ATP energizes ciliary motility and that various cellular signals, including Ca2+, cAMP, and reactive oxygen species, modulate ciliary movements. The precise method for demembranation of C. reinhardtii cells and reactivation of the cell models is described here.

The demembranation and reactivation process in C. reinhardtii wild type strain (CC-125) is shown here. The culture 2 days after inoculation became a light green color (step 1.1) (Figure 1). Cells were collected (step 2.1), washed (step 2.2), and demembranated (step 2.5).&#160;After demembranation, all cell models became immotile (step 2.7). The demembranated cilia (called axonemes) remain attached to the cell body, which shows that the immotility of the cell models is not caused by ...

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Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

The in vitro reactivation of motile cells is a crucial experiment in understanding the mechanisms of cell motility. The protocol describes reactivating the demembranated cell models of Chlamydomonas reinhardtii, a model organism to study cilia/flagella.

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Gy&#246;rgyi demonstrated in the mid-20th century, in ...

reactivation-of-demembranated-cell-models-in-chlamydomonas-reinhardtii

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JoVE Journal

Biology

2.0K Views.  Hosei University. The in vitro reactivation of motile cells is a crucial experiment in understanding the mechanisms of cell motility. The protocol describes reactivating the demembranated cell models of Chlamydomonas reinhardtii, a model organism to study cilia/flagella.

Video: Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

Title Cell Encapsulation by Droplets

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.
Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues 1-6. The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties.
The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type1, 7-13. Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...