野崎久 (東京大学)、仲田崇志 (慶應義塾大学)、由香丸川橋本 (日本女子大学)、関本浩之 (日本女子大学) を感謝いたします。我々 は彼らのコメントの校閲者を感謝したいと思います。本研究はあった科学研究 (号 26440223 ・ 15 H 04413) 日本学術振興会科研費により結城 Tsuchikane に新技術開発財団からの助成金でサポートされています。

1. 藻類を含む水試料の収集
注: 単細胞 conjugatophytes は、湖、湿地、水田など、停滞の淡水地域で育ちます。藻類を含む水のサンプルは、文化のひずみを生成するこれらの環境のいずれかから収集されます。プロセスを開始する前に次のアイテムが必要: 水のサンプルを収集するための 50 mL のチューブやボトル使い捨てピペット プランクトン ネット。
<ol><li>プランクトン湖環境でネットを使用すると、水から藻類を収集します。目的に応じて、適切なネットを使用します。 注: ネットの上部は水を通さないです。ネットでキャッチ藻類プランクトン ネットの下にあるコンテナーに集中しています。粗いメッシュでは、小さな藻類を通過することができます。細かいメッシュは簡単に目詰まりを取得します。</li>
	<li>50 mL チューブまたは 100 mL のペットボトルに水 (約 50 mL) を転送します。研究室に濃縮水のサンプルをもたらします。</li>
	<li>単細胞の conjugatophytes はまた沼地や田んぼで育ちます。これらの浅い水域で藻類を含む水試料からスポイトで直接サンプリングすることができます。その後、50 mL のチューブまたは 50 mL のペットボトルに水 (30-50 mL) を転送します。 注: サンプルに任意の土壌が含まれていないので、水を地表上サンプリング慎重に。</li>
</ol>2 藻類の確認
注: 次の項目が必要です: 観察用ルーペや携帯顕微鏡 60 mm 皿します。
<ol><li>使い捨てのピペットの中にサンプルで藻類の存在を確認するのにには、サンプルを直接観察するのにルーペを使用します。 注: 約より大きいセル 400 1,000 μ m 長さ (例えば、ミカヅキモ ehrenbergii) することができます簡単にこのメソッドを使用して観察されます。</li>
	<li>プラスチック実験室の皿で水サンプルを注ぐポータブル顕微鏡下で藻類を確認します。<ol>
<li>上半分の内側の部分のサンプル (約 3 mL) を入れ (ふた) 60 mm シャーレの上に、蓋の内側の部分に直面してその下の部分で、下半分を入れ。 注: このメソッドは移動するからサンプルを保ち、観察が容易になります。</li>
	</ol>
</li>
</ol>3. パスツール ピペットからガラス毛細管ピペットの隔離のための準備
メモ: プロセスを開始する前に次のアイテムが必要: 滅菌パスツール ピペット、パスツール ピペット、手袋、ゴム球、アルコール ランプ、アルコール ランプ、ライター、鉗子、ガラス用のゴミ箱のメタノールのラックは、クリーン ルームや層流流フード (必要に応じて)。
<ol><li>アルコール バーナーに火を付けます。<ol>
<li>隔離のための細かいガラス毛細管ピペットを生成、炎が点滅していないことを確認します。炎を削るし、その先端にパスツール ピペットを溶かします。</li>
	</ol>
</li>
	<li>熱滅菌ガラス炎、鉗子を使用して手で、先端側をゴム球の側面サポートにパスツール ピペット。</li>
	<li>炎をパスツール ピペットを取り出して、それを伸ばします。<ol>
<li>ガラスを溶かすときは、炎からパスツール ピペットをすばやく削除、引き出します。 注: この実験でパスツール ピペット増築によって 15-20 cm 作る確かピペットは、引く時、炎をオフします。</li>
	</ol>
</li>
	<li>ガラス毛細管ピペットの先端を適切な長さに分割します。湾曲部分が最高級いるので先端に最適です。 注: この研究では 5-10 cm の先端のガラス毛細管ピペットが準備されました。<ol>
<li>必ず、それを溶融後キャピラリーを破棄し、炎を出すことを確認してください。</li>
	</ol>
</li>
	<li>ガラス毛細管ピペットからでる泡は、先端開放のサイズを示します。分離するセルの適切なサイズを選択します。泡の約 1 mm は 10-20 μ m の細胞幅のミカヅキモ属の分離のために適切。</li>
</ol>4 ガラス毛細管ピペットで単一細胞分離
注: 次の項目は開始する前に必要な: 倒立顕微鏡、時計ガラス、プラスチックの皿と滅菌スレッド試験管 CA 媒体 (表 1) を含んでいるまたは CA 中7を完備します。
<ol><li>いくつかの時計メガネ (22 mm 径、深さ 1 mm)、生殖不能の CA 媒体含む各 1 mL を準備します。</li>
	<li>フィールドで採取された水試料から標的細胞を分離します。<ol>
<li>プラスチックの皿に標的細胞を含む水試料 (約 30-40 mL) を注ぎなさい。</li>
		<li>倒立顕微鏡下でプラスチックの皿にサンプルを観察しながらガラス毛細管ピペットを使用して単一セルを拾います。</li>
		<li>ガラス毛細管ピペットを毛管作用によって細胞の吸引を容易にするセルに寄せます。</li>
	</ol>
</li>
	<li>生殖不能の CA 媒体 (図 1 a) 1 mL を含む時計ガラスにセルを転送します。</li>
	<li>生殖不能のカリフォルニア州媒体から新しいガラス毛細管ピペットを使用して再度セルをピックアップし、生殖不能の CA 媒体 (図 1 b) を含む 2 番目の時計ガラスにそれを転送します。 注: 単一のセルはスポット プレート (図 1 c) で分離されているまで、このプロセスは繰り返されます。</li>
	<li>新しいガラス毛細管ピペットを使用して 1 つのセルをピックアップし、最終的にカリフォルニア州媒体の馴化 CA 培地 (図 1 d) 14 mL を含むテスト チューブに転送。23 ° C 16 h 光: 8 の下でサンプルをインキュベート-暗い h は 2 週間サイクルします。 注: 周囲の環境に成長している細胞から分泌される細胞増殖の未知の因子は、細胞分裂4を容易にする条件の中に含まれます。クローン培養が確立される場合培地4からエアコンの CA メディアを準備します。文化は、成功した場合は緑色になるでしょう。</li>
</ol>

<ol>
	<li>Wickett, N. 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=Phylotranscriptomic+analysis+of+the+origin+and+early+diversification+of+land+plants.">Phylotranscriptomic analysis of the origin and early diversification of land plants.</a> Proceedings of the National Academy of Sciences. 111, E4859-E4868 (2014).</li><li>Delwiche, C. F., Cooper, 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=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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+media+for+fresh-water+algae:+problems+and+suggestions+.">Artificial media for fresh-water algae: problems and suggestions .</a> The Ecology of Algae, a symposium held at the Pymatuning Laboratory of Field Biology on June 18 and 19, 1959. , 84-96 (1960).</li><li>Provasoli, L., Tryon, C. A., Hartmann, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Media++&amp;+prospects+for+the+cultivation+of+marine+algae.">Media &amp; prospects for the cultivation of marine algae.</a> Proceedings of U. S.-Japan Conference in Hakone, Japan. , 63-75 (1968).</li><li>Guillard, R. R. L., 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=Purification+methods+for+microalgae.">Purification methods for microalgae.</a> Algal Culturing Techniques: Purification methods for microalgae. , 117-132 (2005).</li></ol>

現在のメソッドを使用して、ミカヅキモsp 9 クローン培養株は 60% の成功率を表す水のサンプル (Inba1) から分離された 15 のセルから確立されました。種の同定は、形態学的観察と分子系統解析8などの DNA 解析によって将来的に実施するでしょう。
提案の方法では、試料水の鮮度は成功率に重要です。フィールド コレクション後できるだけ早く水サンプルから細胞を分離することをお勧めします。細胞の分離が望ましいが、ミカヅキモ属 (例えば c. ehrenbergii c. 学位論文紹介複雑な) セルは 4-8 ° C で保存することによって約 7 d のサンプルを水で維持できる、コレクションまたは次の日の日。また、洗濯の繰り返しの数を増やすので、標的細胞以外の任意の汚染物質を除去することが重要だ (すなわち、> 3 x) 細胞と土壌微生物による文化の汚染を防ぐことができます。
この技法の制限は、このプロトコル (CA または CA の馴化培地) で使用される文化メディア、必ずしもすべて接合藻類の適切です。いくつかのケースでは、他の媒体と同様、異なる光および温度条件の使用を検討する必要があります。また、文化が 1 つのセルから成長しているかどうかを証明することは困難は、テスト チューブにセルを転送するときだけ 1 つのセルを正確に選択する必要が。したがって、転送すべき新しいガラス毛細管ピペットでそれぞれの時間。
このピペット洗浄法とクローン培養を確立するクラシック/標準の方法を研究に必要な細胞を分離に使用する最も信頼性の高い方法です。多く研究8,9,,1011で使用される藻類の活用のクローンの文化は、現在のメソッドを使用して確立されました。クローン培養せず接合藻類を分析することは困難です。また、近年、 de novo全ゲノム シーケンス (例えば、ミニオン ナノポア sequencer を使用して) 取得、やすくなった、 de novoシーケンスがより一層高まるクローン文化を確立します。つまり、このメソッドは、最初のステップは、将来的にこれらの藻類の生物多様性を理解するための研究です。
安定した培養株を確立するためにプロトコル内で正確に特定の重要な手順を実行する必要は。1 つのセルのみの通過を許可する最適な絞り値とガラス毛細管ピペットの準備の最も重要なステップです。ガラス毛細管ピペットの絞りは興味のセルの短軸長さよりわずかに多くである必要があります。最適なガラス毛細管ピペットは、毛管作用によって単一のセルを吸引できます。ただし、開口部の直径が大きすぎる場合、毛細血管は非生活汚染物質材料やも (、) 他嫌気、ターゲット セルに加えて描画もできます。
最適な絞りとガラス毛細管ピペットを得るためには、次の点が注意してください。まず、ガラス毛細管ピペットを加熱するとき、炎の柱が細い必要があります。次に、パスツール ピペットを引っ張って、それは炎; から削除するがそうでなければ、パスツール ピペットの絞りが崩壊します。
機器とこのプロトコルに示すように容器の基本的な変更することができます。たとえば、1 つも時計メガネではなくウェル プレートを使用して、セル洗浄可能より効率的です。さらに、他の類似のものコンテナーを置き換えることが。最後のステップで培養液中に単一のセルを転送する、クローン培養を確立できます。
滅菌コンテナーを準備し、フードでの作業は無菌 (汚染) 系統を確立します。汚染を防ぐためには、3 倍以上の新しい因数洗浄手順を繰り返す必要は。場合によっては、抗生物質15を追加することによって細菌を殺菌することも。
このメソッドは当ててチリモ (Zygnematales) 分離がガラス毛細管ピペット開口部の大きさを変更すると、それは、異なるサイズのプランクトンの分離に適用できます。

(注文 Zygnematales) の藻類の活用、またとして知られている conjugatophytes、占める土地の植物1,2の即時の祖先の最も近い生活親戚として重要な系統位置です。藻類の確立されたクローン文化は、過去年にわたってさまざまな分類学、生理学、遺伝学的研究につながっています。自然のフィールドからの微生物の分離技術は、長い伝統3,4を持っています。近年、フローサイトメトリーを用いた技術はまたずっと自動分離は5を設立しました。クローン培養の確立のための単一のセルを取得するために使用する最も一般的な方法は、ガラス毛細管ピペット6を使用して単一細胞分離です。この従来の方法では、スキルと正確な実験的プロトコルを必要があります。
この記事で示す藻類のサンプリング フィールド サンプルとミカヅキモ種などの単細胞接合藻類の栄養文化の確立からガラス毛細管ピペットを使用して.栄養細胞を含む水試料は、(例えば湖、池、または水田) 現場から収集されます。セルをガラス毛細管ピペットを用いて試料水から分離して洗浄し。16 h 光: 8 の下の 24 の ° C で窒素添加培地 (CA 中) を含む試験管に培養される細胞-h の暗い政権。このメソッドもクローン文化を生成する他の微細藻類を分離するのに最適です。

<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

それは生理学、遺伝学、分類学、微生物学などの様々 なトピックの研究に使用する微細藻類の栄養系文化を確立することが欠かせません。したがって、クローンの文化を確立する技術を開発する非常に重要です。この記事では、抱合藻の栄養文化の確立を示しています。水試料は、フィールドから収集されます。その後、セルはガラス毛細管ピペット、メディアに配置され、クローン培養の生成に適した条件下で栽培を使用して分離されます。

50 mL の水サンプル (Inba1) を 1 つのサンプリング ポイント印旛沼で採取した (pH 8.1 24.7 ° C; 35 ° 44′30. 2″N 140 ° 12′08 です。 2″E) 2016 年 6 月 25 日に千葉県佐倉市に。4-8 ° C で水のサンプルが格納されました。コレクション、ミカヅキモsp の標本が栄養細胞を含む水試料から分離された後の次の日。同一形態の 15 の栄養細胞 (Inba1 1、-2、-3、-4、-5、-6、-7、-8、-9,-10,-11、-12、-13、-14 と-15) サンプルから隔離されピペット洗浄法を使用して洗浄し。15 孤立 1 細胞は CA の馴化培地 15 mL を含む独立した試験管で培養しました。9 試験の試験管内培養 2 週間後細胞増殖が観察された [Inba1-1、-3、-4、-5 (図 2 a)、-6、-9、-10、(図 2 b)、-12、-13;表 2]。9 クローン文化は、Inba1 サンプル分離 (図 3) から確立されました。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57761/57761fig1.jpg"> 図 1: 単一細胞分離の流れ。(、) 元の水のサンプルから、滅菌への単一ターゲット藻類細胞転送中最初の時計ガラス。(b、 c)次の時計ガラスを洗浄するにセルを再び転送します。培地のターゲットより他の細胞/汚染物質がなくなるまで、この手順を繰り返す必要があります。3 つの時計眼鏡は例として図をここに示します。(d) 最後に、成長のための適切な媒体のテスト チューブを洗浄のセルを転送します。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57761/57761fig2v2.jpg"> 図 2: 植物細胞の写真ミカヅキモsp(、) Inba1 5、Inba1-12 (b) に示します。スケールバー = 50 μ m.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig2v2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57761/57761fig3.jpg"> 図 3: Inba1 サンプルからクローン培養の確立します。2 週間培養試験管は、下から撮影されました。アスタリスクは、細胞増殖を示します。スケール バー = 2 cm<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57761/57761fig3large.jpg" target="_blank">この図の拡大版を表示するにはここをクリックしてください。</a> 。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>メディアの名前</td>
			<td>参照</td>
			<td colspan="2">コメント</td>
		</tr>
<tr>
<td rowspan="13">CA</td>
			<td rowspan="13">市村と渡辺12
</td>
			<td>Ca (3)2·4H2O</td>
			<td>2 mg</td>
		</tr>
<tr>
<td>自演3
</td>
			<td>10 mg</td>
		</tr>
<tr>
<td>NH43
</td>
			<td>5 mg</td>
		</tr>
<tr>
<td>Β-Na2glycerophosphate·5H2O</td>
			<td>3 mg</td>
		</tr>
<tr>
<td>MgSO4·7H2O</td>
			<td>2 mg</td>
		</tr>
<tr>
<td>ビタミン B12</td>
			<td>0.01 μ g</td>
		</tr>
<tr>
<td>ビオチン</td>
			<td>0.01 μ g</td>
		</tr>
<tr>
<td>チアミン HCl</td>
			<td>1 μ g</td>
		</tr>
<tr>
<td>PIV 金属</td>
			<td>0.1 mL</td>
		</tr>
<tr>
<td>Fe (EDTA; として 1:1 大臼歯)</td>
			<td>0.1 mL</td>
		</tr>
<tr>
<td>HEPES</td>
			<td>40 mg</td>
		</tr>
<tr>
<td colspan="2">100 mL を作るために水を追加します。</td>
		</tr>
<tr>
<td colspan="2">7.2 NaOH で pH を調整します。</td>
		</tr>
<tr>
<td rowspan="7">P IV 金属</td>
			<td rowspan="7">Provasoli と Pintner の13
</td>
			<td>ナ2EDTA·2H2O</td>
			<td>100 mg</td>
		</tr>
<tr>
<td>した FeCl3·6H2O</td>
			<td>19.6 mg</td>
		</tr>
<tr>
<td>MnCl2·4 H2O</td>
			<td>3.6 mg</td>
		</tr>
<tr>
<td>ZnCl2*</td>
			<td>1.04 mg</td>
		</tr>
<tr>
<td>CoCl2·6H2O</td>
			<td>0.4 mg</td>
		</tr>
<tr>
<td>ナ2MoO4·2H2O</td>
			<td>0.25 mg</td>
		</tr>
<tr>
<td colspan="2">100 mL を作るために水を追加します。</td>
		</tr>
<tr>
<td rowspan="3">Fe (EDTA; として 1:1 大臼歯)</td>
			<td rowspan="3">Provasoli14
</td>
			<td>Fe (NH4)2(その4)2·6H2O、</td>
			<td>70.2 mg</td>
		</tr>
<tr>
<td>Na2EDTA·2H2O、</td>
			<td>66 mg</td>
		</tr>
<tr>
<td colspan="2">100 mL を作るために水を追加します。</td>
		</tr>
<tr>
<td>CA の馴化培地</td>
			<td>阿部ら7
</td>
			<td colspan="2">14-20 日間の新鮮な CA の培地で細胞を孵化させなさい。定性ろ紙を使用してろ過で培地を収集し、オートクレーブ滅菌 (121 ° C で 15 分) によってフィルター処理された培地を殺菌しなさい。</td>
		</tr>
<tr>
<td colspan="4">* で、nies 向け 1.04 mg ZnCl2に置き換えは 2.2 mg ZnSO4·7H2o.</td>
		</tr>
</tbody></table>本研究で使用されるメディアの表 1: 配合。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>水のサンプル</td>
			<td>セルの名前</td>
			<td>培養後の状態</td>
			<td>緊張の名前</td>
		</tr>
<tr>
<td rowspan="15">Inba1</td>
			<td>-1</td>
			<td>細胞増殖</td>
			<td>Inba1 1</td>
		</tr>
<tr>
<td>-2</td>
			<td>変更はできません。</td>
			<td></td>
		</tr>
<tr>
<td>-3</td>
			<td>細胞増殖</td>
			<td>inba1 3</td>
		</tr>
<tr>
<td>-4</td>
			<td>細胞増殖</td>
			<td>inba1 4</td>
		</tr>
<tr>
<td>-5</td>
			<td>細胞増殖</td>
			<td>inba1 5</td>
		</tr>
<tr>
<td>-6</td>
			<td>細胞増殖</td>
			<td>inba1 6</td>
		</tr>
<tr>
<td>-7</td>
			<td>変更はできません。</td>
			<td></td>
		</tr>
<tr>
<td>-8</td>
			<td>変更はできません。</td>
			<td></td>
		</tr>
<tr>
<td>-9</td>
			<td>細胞増殖</td>
			<td>inba1 9</td>
		</tr>
<tr>
<td>-10</td>
			<td>細胞増殖</td>
			<td>inba1 10</td>
		</tr>
<tr>
<td>-11</td>
			<td>変更はできません。</td>
			<td></td>
		</tr>
<tr>
<td>-12</td>
			<td>細胞増殖</td>
			<td>inba1 12</td>
		</tr>
<tr>
<td>-13-</td>
			<td>細胞増殖</td>
			<td>inba1 13</td>
		</tr>
<tr>
<td>-14</td>
			<td>変更はできません。</td>
			<td></td>
		</tr>
<tr>
<td>-15</td>
			<td>変更はできません。</td>
			<td></td>
		</tr>
</tbody></table>表 2: 分離試験概要。

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

抱合の単細胞藻類の栄養文化の確立

この記事では、自然の現場から採集した単細胞の抱合藻類種のクローンの文化の確立を示しています。

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 ビュー.  Japan Women's University. この記事では、自然の現場から採集した単細胞の抱合藻類種のクローンの文化の確立を示しています。

ビデオ: 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.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

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

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.

クローン性増殖のためのCD133 +肝幹細胞の単離

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.

新しい多層フラスコを用いて哺乳動物細胞培養のスケールアップ

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.

化学的に成層南極湖と炭素固定ポテンシャルの評価から微生物真核生物濃縮文化の確立

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.

外植片培養での歯胚の開発に続いてのためのスライス培養法

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.

植物や緑藻の光捕集複合体のin vitro再構成では、

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.

酸性細胞外 pH 培養系の確立

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.

グノトビオティック・アメリカン・ゴキブリの設立と維持 (ペリプラネタ・アメリカーナ)

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.

ウシ卵巣皮質組織培養

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.