我们感谢 Hisayoshi 野崎 (东京大学)、隆 Nakada (京庆大学)、原田 Marukawa 桥本 (日本女子大学) 和博行 Sekimoto (日本女子大学)。我们要感谢评价员的意见。这项研究得到了日本科学促进协会 (26440223 号和 15H04413) 资助的资助, 并得到了来自新科技发展基金会的赠款, Tsuchikane。

1. 收集含有藻类的水样
注: 单细胞 conjugatophytes 生长在停滞的淡水地区, 如湖泊、沼泽和稻田。从这些环境之一中收集含藻类的水样, 以产生一种培养菌株。在开始该过程之前需要下列项目: 浮游生物网, 一次性吸管, 和一个瓶子或50毫升管收集水样。
<ol><li>在湖泊环境中使用浮游生物网从水中收集藻类。根据用途, 使用适当的网。 注意: 网的上部对水是透水的。被网捕获的藻类集中在浮游生物网的底部容器中。一个粗糙的网格允许小藻类通过。细网容易被堵塞。</li>
	<li>将水 (约50毫升) 转移到50毫升管或100毫升塑料瓶。把浓缩的水样带到实验室。</li>
	<li>单细胞 conjugatophytes 也生长在沼泽或稻田。在这些浅水区域, 含有藻类的水样可以直接用吸管取样。然后, 将水 (30-50 毫升) 转移到50毫升管或50毫升塑料瓶。 注: 水仔细取样在土壤表面之上, 以便样品不包含任何土壤。</li>
</ol>2. 藻类的确认
注意: 以下项目是必需的: 放大镜或便携显微镜和60毫米的盘子观察。
<ol><li>为了确认样品中藻类存在于一次性吸管中, 请使用放大镜直接观察样品。 注意: 使用此方法可以很容易地观察到大约 400-1000 µm 的较大细胞 (例如,新月 ehrenbergii)。</li>
	<li>在便携式显微镜下确认藻类, 将水样倒入塑料实验盘中。<ol>
<li>将样品 (约3毫升) 放在60毫米培养皿的上半部分 (盖子) 的内侧, 然后将下半部分放在下部, 其底部朝向盖子的内部, 上面。 注: 此方法使样品不动, 使观察更容易。</li>
	</ol>
</li>
</ol>3. 从巴斯德吸管中制备玻璃毛细管吸管隔离
注意: 在开始该过程之前, 需要下列项目: 灭菌的巴斯德吸管, 一架为巴斯德吸管, 手套, 橡胶灯泡, 精神灯, 甲醇为精神灯, 打火机, 镊子, 垃圾桶为玻璃, 和一个干净的房间或层流流动罩 (如有必要)。
<ol><li>点燃酒精燃烧器。<ol>
<li>要生产一个细玻璃毛细管隔离, 确保火焰没有闪烁。锐化火焰, 并在其尖端熔化巴斯德吸管。</li>
	</ol>
</li>
	<li>用镊子在火焰上加热无菌玻璃巴斯德吸管, 用手和笔尖在橡胶灯泡一侧支承。</li>
	<li>把巴斯德吸管脱下火焰, 伸展它。<ol>
<li>当玻璃融化时, 迅速从火焰中取出巴斯德吸管, 然后拉。 注: 在本实验中, 巴斯德吸管的扩展为 15-20 厘米. 在拉拔过程中, 要确保吸管脱离火焰。</li>
	</ol>
</li>
	<li>将玻璃毛细管吸管的尖端折断到适当的长度。因为鞠躬的部分是最好的, 它是理想的尖端。 注: 在本研究中, 制备了一个 5-10 厘米尖端的玻璃毛细管吸管。<ol>
<li>在熔化后一定要丢弃毛细管, 并确认火焰熄灭。</li>
	</ol>
</li>
	<li>从玻璃毛细管吸管释放出来的气泡表明尖端开口的大小。选择要隔离的单元格的适当大小。大约1毫米气泡是适当的分离新月物种, 与细胞宽度 10-20 µm。</li>
</ol>4. 用玻璃毛细管吸管分离单细胞
注: 在启动前需要下列物品: 倒置光镜、手表眼镜、塑料碟和含钙介质的无菌螺纹试管 (表 1) 或条件 ca 中等7。
<ol><li>准备一些手表眼镜 (直径22毫米和1毫米深度), 每只含有1毫升的无菌钙培养基。</li>
	<li>将目标单元格与在该字段中收集的水样样本隔离。<ol>
<li>将含有靶细胞的水样 (约 30-40 毫升) 倒入塑料盘中。</li>
		<li>在用倒置的光镜观察塑料碟中的样品时, 用玻璃毛细管吸管提取单细胞。</li>
		<li>在细胞附近带上玻璃毛细管吸管, 用毛细管作用来促进细胞的吸入。</li>
	</ol>
</li>
	<li>将细胞转移到含有1毫升无菌 CA 培养基的手表玻璃上 (图 1a)。</li>
	<li>再用新的玻璃毛细管从不育的钙介质中提取细胞并将其转移到含有无菌 ca 介质的第二个手表玻璃上 (图 1b)。 注意: 此过程是重复的, 直到单个单元格被隔离在专版中 (图 1c)。</li>
	<li>使用新的玻璃毛细管吸管拾取单细胞, 最后将其转移到包含14毫升 ca 介质或条件 ca 介质的试管中 (图 1d)。在16小时 light:8-h 黑暗周期的2周内, 将样品孵化23摄氏度。 注: 细胞增殖的未知因素, 从生长细胞分泌到周围环境中, 包括在条件培养基中以促进细胞分裂4。如果建立克隆文化, 则从培养基4中制备条件 CA 培养基。如果成功, 文化将变成绿色。</li>
</ol>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+origin+of+a+terrestrial+flora.">The evolutionary origin of a terrestrial flora.</a> Current Biology. 25, R899-R910 (2015).</li><li>Daste, P., Neuville, D., Victor-Baptiste, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+obtaining+clonal+isolation+of+diatoms.">A simple procedure for obtaining clonal isolation of diatoms.</a> British Journal of Psychology. 18, 1-3 (1983).</li><li>Pringsheim, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pure Cultures of Algae: Their Preparation and Maintenance. , 119 (1946).</li><li>Sieracki, M., Poulton, N., Crosbie, N., Andersen, 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=Automated+isolation+techniques+for+microalgae.">Automated isolation techniques for microalgae.</a> Algal Culturing Techniques: Automated isolation techniques for microalgae. , 101-116 (2005).</li><li>Andersen, R. A., Kawachi, M., Andersen, 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=Traditional+microalgae+isolation+techniques.">Traditional microalgae isolation techniques.</a> Algal Culturing Techniques: Traditional micro algae isolation techniques. , 83-100 (2005).</li><li>Abe, 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=Stable+nuclear+transformation+of+the+Closterium+peracerosum-strigosum-littorale+complex.">Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex.</a> Plant and Cell physiology. 52, 1676-1685 (2011).</li><li>Tsuchikane, Y., Tsuchiya, M., Kokubun, Y., Abe, J., Sekimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+processes+of+Penium+margaritaceum+(Zygnemophyceae,+Charophyta).">Conjugation processes of Penium margaritaceum (Zygnemophyceae, Charophyta).</a> Phycological Research. 59, 74-82 (2011).</li><li>Kanda, N., 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=CRISPR/Cas9-based+knockouts+reveal+that+CpRLP1+is+a+negative+regulator+of+the+sex+pheromone+PR-IP+in+the+Closterium+peracerosum-strigosum-littorale+complex.">CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.</a> Scientific Reports. 7, 17873 (2017).</li><li>Ichimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+types+and+reproductive+isolation+in+Closterium-ehrenbergii+Meneghini.">Mating types and reproductive isolation in Closterium-ehrenbergii Meneghini.</a> Botanical Magazine-Tokyo. 94, 325-334 (1981).</li><li>Sekimoto, H., Satoh, S., Fujii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+physiological+properties+of+a+protein+inducing+protoplast+release+during+conjugation+in+the+Closterium+peracerosum-strigosum-littorale+complex.">Biochemical and physiological properties of a protein inducing protoplast release during conjugation in the Closterium peracerosum-strigosum-littorale complex.</a> Planta. 182, 348-354 (1990).</li><li>Ichimura, T., Watanabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Closterium+calosporum+complex+from+the+Ryukyu+Islands-Variation+and+taxonomical+problems.">The Closterium calosporum complex from the Ryukyu Islands-Variation and taxonomical problems.</a> Memoirs of the National Science Museum. 7, (1974).</li><li>Provasoli, L., Pintner, I. 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采用现有的方法, 从水样中分离出的15个细胞 (Inba1) 中, 建立了9株新月无性系培养菌株, 成功率达60%。今后将通过形态学观察和 DNA 分析等方法对物种进行鉴定, 如分子系统发育分析8。
在本方法中, 水样的新鲜度对成功率有重要意义。在野外采集后, 最好尽快将细胞从水样中分离出来。虽然新月物种的细胞 (例如, ehrenbergii, peracerosum-strigosum 护胸复合物) 可以保存在水样品约 7 d, 通过储存在 4-8 摄氏度, 细胞的隔离是可取的在收集的当天或第二天。除去目标细胞以外的任何污染物也很重要, 因此增加洗涤重复次数 (即> 3x) 可以防止土壤和细胞中的微生物对培养物造成污染。
这种技术的一个局限性是, 本协议中使用的培养基 (ca 或条件 ca 培养基) 并不总是适合所有的共轭藻类。在某些情况下, 可能需要考虑使用另一种介质, 以及不同的光和温度条件。此外, 由于很难证明某种文化是否从一个细胞中生长出来, 所以在将细胞转移到试管时, 必须准确地提取出1个细胞。因此, 每次都要用新的玻璃毛细管吸管进行转移。
用这种吸管洗涤法建立克隆文化是一种经典/标准方法, 是用来分离所需细胞进行研究的最可靠的方法。采用本方法建立了多项研究8、9、10、11共轭藻类的无性系培养。没有克隆文化, 很难分析共轭藻类。此外, 近年来,从头开始的整个基因组序列变得容易获得 (例如, 通过使用奴才纳米排序器), 并建立克隆文化将进一步促进新的排序。换言之, 这种方法是今后研究这些藻类的第一步, 以更好地了解它们的生物多样性。
为了建立稳定的文化张力, 必须在协议中准确地执行某些关键步骤。最重要的步骤是制备一个具有最佳孔径的玻璃毛细管吸管, 仅允许单个细胞通过。玻璃毛细管吸管的孔径必须略大于所感兴趣细胞的小轴长度。最佳的玻璃毛细管吸管可以通过毛细管作用吸入单个细胞。然而, 如果孔径的直径太大, 毛细管也将绘制非生物污染材料, 甚至 (a) 其他有机体, 除了目标细胞。
要获得最佳孔径的玻璃毛细管吸管, 必须注意以下几点。首先, 在加热玻璃毛细管时, 火焰的柱必须是狭窄的。其次, 当拔出巴斯德吸管时, 必须从火焰中除去它;否则, 巴斯德吸管的光圈就会崩溃。
本协议中显示的设备和容器是基本的, 可以修改。例如, 通过使用多孔板而不是用一个井观察眼镜, 可以使细胞洗涤更有效率。此外, 集装箱可以替代其他类似的。在最后一步, 通过将单个细胞转移到培养基上, 可以建立克隆培养。
准备一个灭菌容器和工作在一个罩将建立无菌 (无污染) 菌株。为了防止污染, 有必要重复清洗步骤与新鲜整除数超过3x。有时, 细菌也通过添加抗生素15消毒。
该方法侧重于 desmid (Zygnematales) 隔离, 但通过改变玻璃毛细管孔径的大小, 可以将其应用于不同大小的浮游生物的分离。

共轭藻类 (顺序 Zygnematales), 也被称为 conjugatophytes, 占据关键的系统发育地位, 作为最接近的生活亲属的直接祖先的土地植物1,2。在过去的几年里, 建立了藻类的克隆文化, 导致了各种各样的分类学、生理和遗传学研究。微生物的分离技术从一个自然领域有一个长的传统3,4。近年来, 采用流式细胞仪的自动隔离技术也建立了5。最常用的方法, 以获得一个单一的细胞建立克隆文化是单细胞隔离使用玻璃毛细管吸管6。这种传统的方法需要技巧和精确的实验协议。
在本文中, 我们展示了从田间样品中藻类的取样和建立单细胞共轭藻类的克隆文化, 如新月物种, 使用玻璃毛细管吸管。一个含有营养细胞的水样本是从一个田野遗址 (如湖泊、池塘或稻田) 收集的。细胞与水样分离, 使用玻璃毛细管吸管, 然后清洗。细胞是培养在一个试管中含有氮补充培养基 (CA 培养基) 在24摄氏度下, 在一个16小时 light:8-h 黑暗政权。该方法也适用于分离其他微藻生成无性系培养。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sampling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plankton net</td>
			<td>RIGO, Japan</td>
			<td>N-NO380T</td>
			<td>Mesh size: 32 &#181;m</td>
		</tr>
		<tr>
			<td>Portable microscope (e.g., Nature Scope FABRE)</td>
			<td>Nikon, Japan</td>
			<td>JAN: 4960759 206725</td>
			<td>Magnification: 20&#215;</td>
		</tr>
		<tr>
			<td>Loupe (e.g., Peak 1985 Steinheil System Magnifier)</td>
			<td>Tohkai Sangyo Co. Ltd., Japan</td>
			<td>1985-20</td>
			<td>Magnification: 20&#215;</td>
		</tr>
		<tr>
			<td>Spuit</td>
			<td>EIKEN CHEMICAL CO. LTD. Japan</td>
			<td>Sterile spuit No.4</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Tube (50 mL centrifuge tubes with screw caps)</td>
			<td>Labcon, USA</td>
			<td>3181-345-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle (100 mL Polycarbonate bottle)</td>
			<td>AS ONE Corporation, Japan</td>
			<td>1-7403-01</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm dish</td>
			<td>Asahi glass Co. Ltd., Japan</td>
			<td>1010-060</td>
			<td>&#160;For observation of algae.&#160; 60 mm/non-treated dish</td>
		</tr>
		<tr>
			<td>Preparation of a micropipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (cotton plugged 9&#8221; Pasteur Pipettes)</td>
			<td>Fisher Scientific, USA</td>
			<td>13-678-8B</td>
			<td>7 &#215; 225 mm</td>
		</tr>
		<tr>
			<td>Rubber bulb (SPOID SILICONE, 1 cc)</td>
			<td>AS ONE Corporation, Japan</td>
			<td>5-5669-01</td>
			<td>10 &#215; 40 mm</td>
		</tr>
		<tr>
			<td>Stainless forceps</td>
			<td>AS ONE Corporation, Japan</td>
			<td>PT-09</td>
			<td>110 mm</td>
		</tr>
		<tr>
			<td>Single-cell isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass (Blood reaction board)</td>
			<td>Sekiya Rika Co., Ltd, Japan</td>
			<td>F14-155-030</td>
			<td>to rinse cells during isolation. 22 mm diameter and 1 mm depth</td>
		</tr>
		<tr>
			<td>Threaded test tubes&#160;</td>
			<td>Fujimotorika, Co., Ltd, Japan</td>
			<td>XX142</td>
			<td>18 &#216; &#215; 170 mm</td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>CKX41N</td>
			<td>for isolation of algae.</td>
		</tr>
		<tr>
			<td>Plastic dish</td>
			<td>Asahi glass Co. Ltd., Japan</td>
			<td>SH90-15E&#160;</td>
			<td>&#160;90 &#215; 15 mm</td>
		</tr>
	</tbody>
</table>

establishment of a clonal culture of unicellular conjugating algae

必须建立微藻的克隆文化, 用于研究各种主题, 如生理学、遗传学、分类学和微生物学。因此, 建立克隆文化的技术极为重要。本文展示了共轭藻无性系培养的建立。从田间采集水样。随后, 细胞被隔离使用玻璃毛细管吸管, 放置在媒体, 并生长在条件适合生成克隆文化。

在 2016年6月25日, 日本千叶樱花石 (Inba) 的一个取样点 (pH 8.1, 24.7 °c; 35°44′30.2″N 140°12′08.2″E) 采集了50毫升的水样 (Inba1)。水样品被存放在 4-8 °c。第二天收集后,新月的标本从含有营养细胞的水样中分离出来。十五相同形态的营养细胞 (Inba1-1,-2、-3、-4、5、-6、-7、-8、-9、-10、-11、-12、-13、-14、15、) 与样品分离, 然后使用吸管洗涤法进行清洗。在含有15毫升条件的钙培养基的独立试管中培养出十五株孤立单细胞。经过2周的孵化, 在9试管中观察到细胞增殖 [Inba1-1,-3,-4,-5 (图 2a),-6,-9,-10,-12 (图 2b), 和-13;表 2]。从 Inba1 样本分离株中建立了九种克隆培养物 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57761/57761fig1.jpg"> 图 1: 单细胞分离的流动.(a) 将单一目标藻类细胞从原水样品转移到第一个观察玻璃中的无菌介质。(b、 c)再次将细胞转移到下一个手表玻璃上清洗。该程序应重复, 直到没有其他细胞/污染物比目标的媒介;三手表眼镜显示在图这里作为一个例子。(d) 最后, 将冲洗过的电池转移到适当培养基的试管中进行生长。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57761/57761fig2v2.jpg"> 图 2: 植物细胞的照片新月sp.(a) Inba1-5 和 (b) Inba1-12 显示在这里。刻度条 = 50 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig2v2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57761/57761fig3.jpg"> 图 3: 从 Inba1 样品中建立克隆文化.从底部拍摄了2周培养的试管。星号表示细胞增殖。刻度条 = 2 厘米.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>媒体名称</td>
			<td>参考</td>
			<td colspan="2">评论</td>
		</tr>
<tr>
<td rowspan="13">约</td>
			<td rowspan="13">市村和渡边12
</td>
			<td>Ca (NO3)2· 4H2O</td>
			<td>2毫克</td>
		</tr>
<tr>
<td>3
</td>
			<td>10毫克</td>
		</tr>
<tr>
<td>NH4NO3
</td>
			<td>5毫克</td>
		</tr>
<tr>
<td>β–Na2glycerophosphate·5H2O</td>
			<td>3毫克</td>
		</tr>
<tr>
<td>MgSO4· 7H2O</td>
			<td>2毫克</td>
		</tr>
<tr>
<td>维生素 B12</td>
			<td>0.01 微克</td>
		</tr>
<tr>
<td>素</td>
			<td>0.01 微克</td>
		</tr>
<tr>
<td>盐酸硫胺</td>
			<td>1微克</td>
		</tr>
<tr>
<td>PIV 金属</td>
			<td>0.1 毫升</td>
		</tr>
<tr>
<td>Fe (EDTA; 1:1 摩尔)</td>
			<td>0.1 毫升</td>
		</tr>
<tr>
<td>HEPES</td>
			<td>40毫克</td>
		</tr>
<tr>
<td colspan="2">加水使100毫升</td>
		</tr>
<tr>
<td colspan="2">pH 值与氢氧化钠调节为7。2</td>
		</tr>
<tr>
<td rowspan="7">P IV 金属</td>
			<td rowspan="7">Provasoli 和 Pintner13
</td>
			<td>Na2EDTA·2H2O</td>
			<td>100毫克</td>
		</tr>
<tr>
<td>FeCl3· 6H2O</td>
			<td>19.6 毫克</td>
		</tr>
<tr>
<td>MnCl2·4H2O</td>
			<td>3.6 毫克</td>
		</tr>
<tr>
<td>ZnCl2*</td>
			<td>1.04 毫克</td>
		</tr>
<tr>
<td>CoCl2· 6H2O</td>
			<td>0.4 毫克</td>
		</tr>
<tr>
<td>Na2MoO4· 2H2O</td>
			<td>0.25 毫克</td>
		</tr>
<tr>
<td colspan="2">加水使100毫升</td>
		</tr>
<tr>
<td rowspan="3">Fe (EDTA; 1:1 摩尔)</td>
			<td rowspan="3">Provasoli14
</td>
			<td>Fe (NH4)2(如此4)2· 6H2O,</td>
			<td>70.2 毫克</td>
		</tr>
<tr>
<td>Na2EDTA·2H2O,</td>
			<td>66毫克</td>
		</tr>
<tr>
<td colspan="2">加水使100毫升</td>
		</tr>
<tr>
<td>条件 CA 介质</td>
			<td>亚伯等7
</td>
			<td colspan="2">在新鲜的钙培养基中孵化细胞14–20天。用质滤纸过滤培养基, 通过热处理 (121 °c 15 分钟) 对过滤介质进行消毒。</td>
		</tr>
<tr>
<td colspan="4">* 在新兴工业化中, 1.04 毫克 ZnCl2被2.2 毫克 ZnSO4· 7H2O 取代。</td>
		</tr>
</tbody></table>表 1: 本研究使用的媒介配方。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>水样</td>
			<td>单元格名称</td>
			<td>文化后的状态</td>
			<td>应变名称</td>
		</tr>
<tr>
<td rowspan="15">Inba1</td>
			<td>-1</td>
			<td>细胞增殖</td>
			<td>Inba1-1</td>
		</tr>
<tr>
<td>-2</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-3</td>
			<td>细胞增殖</td>
			<td>inba1-3</td>
		</tr>
<tr>
<td>-4</td>
			<td>细胞增殖</td>
			<td>inba1-4</td>
		</tr>
<tr>
<td>-5</td>
			<td>细胞增殖</td>
			<td>inba1-5</td>
		</tr>
<tr>
<td>-6</td>
			<td>细胞增殖</td>
			<td>inba1-6</td>
		</tr>
<tr>
<td>-7</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-8</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-9</td>
			<td>细胞增殖</td>
			<td>inba1-9</td>
		</tr>
<tr>
<td>-10</td>
			<td>细胞增殖</td>
			<td>inba1-10</td>
		</tr>
<tr>
<td>-11</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-12</td>
			<td>细胞增殖</td>
			<td>inba1-12</td>
		</tr>
<tr>
<td>-13</td>
			<td>细胞增殖</td>
			<td>inba1-13</td>
		</tr>
<tr>
<td>-14</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-15</td>
			<td>未更改</td>
			<td></td>
		</tr>
</tbody></table>表 2: 隔离试验摘要。

Watch this Scientific Journal Video about Establishment of a Clonal Culture of Unicellular Conjugating Algae at JoVE.com

Establishment of a Clonal Culture of Unicellular Conjugating Algae

单细胞共轭藻类无性系培养的建立

在这篇文章中, 我们展示了建立的克隆文化的单细胞共轭藻类物种收集从一个自然的现场。

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

establishment-of-a-clonal-culture-of-unicellular-conjugating-algae

Nanyang Technological University

Research

JoVE Journal

Biology

13.1K 次观看.  Japan Women's University. 在这篇文章中, 我们展示了建立的克隆文化的单细胞共轭藻类物种收集从一个自然的现场。

视频: Establishment of a Clonal Culture of Unicellular Conjugating Algae

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

分离的CD133 +肝干细胞的克隆扩增

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

哺乳动物细胞培养，使用一种新的多层瓶中的比例增加

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

建立真核微生物的富集培养从化学分层南极湖和固碳潜力评估

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

在植物和绿藻的捕光配合体外重组

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

牛卵巢皮质组织培养

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.