東京大学先端研、医学システム生物学、ゲノム科学部門と研究室のメンバーに感謝します我々 は特に感謝博士 h. 油谷、小玉徹博士、(LSBM、東大先端研、東京大学)、A 博士、博士・吉田先生・ k ・富塚 有用な議論とサポートの國里 (協和発酵キリン株式会社)。この作品は一部文部の若い科学者 (A) (26710005、ティー ・ オーエンタテインメント)、革新的な領域 (H 01567、ティー ・ オーエンタテインメントの 26116711 と 16) 科学研究費補助金・挑戦 文部科学省がん研究事業がん研究 (ティー ・ オーエンタテインメント)、小林財団、武田科学振興財団 (ティー ・ オーエンタテインメント) 日本の技術からの探索的研究 (16 K 14605、ティー ・ オーエンタテインメント) と(P 作成) 治療の進化とアメッド (ティー ・ オーエンタテインメント) 総合医療研究機構から革新的ながん対策の実践的研究。

1. 酸性の培地の準備
<ol><li>
コントロール (pH 7.4) の作製と低 pH (ペーハー 6.8) 培
	<ol>
<li>500 mL の水に L-グルタミンなし DMEM 粉末 4.75 g を溶解します。</li>
		<li>耐圧ボトルで 0.33 M NaHCO3ソリューションを準備します。 注意: NaHCO3 CO2ガスを加熱すると熱分解を発するので圧力のタイトなボトルを使用することが重要です。</li>
		<li>オートクレーブ媒体およびソリューション。25 ° C に冷却するこれらのバッファーを許可します。</li>
		<li>200 mM L-グルタミン、ペニシリン-ストレプトマイシンの 5 mL の 5 mL を加えると炭酸なし DMEM の 500 mL に牛胎児血清の 50 mL。</li>
		<li>コントロール (pH 7.4) 中 2.4 mL 炭酸なし DMEM の 100 mL あたり 0.33 M NaHCO3ソリューションを追加します。低 pH (ペーハー 6.8) 中炭酸なし DMEM の 100 mL あたり 0.33 M NaHCO3溶液 0.6 mL を追加します。</li>
		<li>37 ° C で 24 時間後 5% 以下の培地の pH をチェック CO2。 注: NaHCO3溶液の量は、インキュベーターと空気圧の CO2の含有量によって異なることが培地の pH に応じて調整する必要があります。理想的には、初めて、DMEM に追加する NaHCO3ソリューションの適切な量を決定する重炭酸塩濃度と培地の pH を測定することをお勧めします。<ol>
<li>CO2インキュベーターから培養皿を削除した直後に培養液を収集し、pH 計を用いて pH を測定します。必要に応じて水浴を使用して 37 ° C で中の温度を保ちます。測定培地の pH 3 回独立。 注: NaHCO3ソリューションは 4 ° C で保存する必要があります、各メディアが新たに作成される必要があります。本研究で使用できる市販の製品は、材料のテーブルに表示されます。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
乳酸誘導または HCl による酸性培養培地の準備
	<ol>
<li>(1.1.5 の手順で準備) 制御媒体の 100 mL に乳酸溶液の 74 μ L または 1 M HCl の 125 μ L を追加します。</li>
		<li>37 ° C で 24 時間後 5% 以下の培地の pH をチェック CO2。 注: 乳酸または塩酸溶液の量はまたインキュベーターと空気圧で CO2コンテンツによって異なることが培地の pH に応じて調整する必要があります。理想的には、シリアルの乳酸や媒体の目的の pH に調整するための適切な量を決定する塩酸濃度と培地の pH を測定することをお勧めします。</li>
	</ol>
</li>
</ol>2. 収穫とセルを扱う
<ol><li>37 ° C 未満 5% CO2を実行する前に、少なくとも 1 週間の実験で細胞の育成を開始します。 注: この研究で使用される細胞は、材料表に表示されます。</li>
	<li>70-90% の confluency に到達するときにセルを通過。コンフルエントになるため細胞を認めない。毎日培養中に 2-3 日毎に媒体を変更します。</li>
	<li>5 mL の PBS を追加することによって、細胞を洗います。PBS を吸引し、1 mL のトリプシンなど脱着酵素を追加します。</li>
	<li>セル丸められますをデタッチする開始になるまでは、37 ° C で孵化させなさい。</li>
	<li>剥離細胞に培養液 5 mL を追加し、酵素を非アクティブ化します。15 mL チューブ 300 x g 吸引上清に 3 分間遠心し、細胞懸濁液を転送、再培養培地で細胞を中断します。</li>
	<li>検定とトリパン ブルーを使用して実行可能なセルを数えます。</li>
	<li>種 5.0 × 105セル/10 mL の培地に 10 センチメートルの皿の井戸。37 ° C 未満 5% で 24 時間細胞をインキュベート CO2。</li>
	<li>培養培地を吸引、5 ml の PBS、細胞を洗浄し、セクション 1 で作製した酸性培地の 10 mL を加えます。37 ° C 未満 5% で 24 時間細胞をインキュベート CO2。</li>
</ol>3. RNA の隔離
<ol><li>培地を吸引し、5 ml の PBS セルを洗ってください。RNA 抽出試薬を追加してセルを破壊 (教材の一覧表を参照してください)。</li>
	<li>料理を簡単にこすり、ピペットと RNA 抽出試薬を削除し、RNA 抽出試薬/細胞ライセートを 1.5 mL チューブを入金します。5 分間室温に置いたまま。</li>
	<li>250 μ L のクロロホルムを加え、5 分間室温で約 15 米残すと ≥8、5 分 000 × g で遠心管を積極的に振る。</li>
	<li>ピペットを使用して水相を慎重に取り外します。水様段階のいくつかの後ろに残します。別 1.5 mL チューブに配置します。</li>
	<li>水相に 550 μ L のイソプロパノールを加え、軽く混ぜます。室温で 5 分間遠心分離機 (≥20, 000 x g) 最大速度、4 ° C で 20 分を残してください。</li>
	<li>イソプロパノールを注ぐし、1 mL 75% エタノール DEPC 処理水を追加します。優しく混ぜます。5 分間 8,000 × g で遠心分離機します。</li>
	<li>ペレットが空気乾燥させエタノールをピペットします。</li>
	<li>RNA ペレットを約 15-25 μ (収量) に応じて DEPC 処理水を追加します。ミックス徹底的。</li>
	<li>260 で外径を測定することによって隔離された RNA 濃度を測定分光光度計を用いた nm。</li>
</ol>4. cDNA 合成
<ol><li>PCR の反作用の管の次のコンポーネントを組み合わせて: 最大 5 μ g 総 RNA オリゴ dT 1 μ L (50 μ M) やランダム プライマー ミックス (60 μ M)、1 μ L 10 mM dNTP、ヌクレアーゼ フリー水 14 μ L の容量を。</li>
	<li>ミックスし、簡単に小さなベンチトップ遠心分離機を使用してコンポーネントを遠心分離機 (~ 5 s)。65 ° C で 5 分間サンプル RNA のプライマーを変化させなさい。簡単にスピン (~ 5 s) 遠心し、少なくとも 1 分間氷の上速やかにサンプルを置く小さな卓上型を使用します。</li>
	<li>次のコンポーネントを追加: 4 μ L 5 x を逆転写バッファー、1 μ L 逆転写酵素 (200 U/μ L)、1 μ L RNase 阻害剤 (40 U/μ L)。 注: 市販キットの例は材料表のとおり
</li>
	<li>チューブをキャップ ミックス、および卓上型小型遠心分離機を使用してコンテンツを簡単に遠心分離機 (~ 5 s)。10 分間で 50-55 ° C 複合反応混合物を孵化させなさい。</li>
	<li>10 分の 80 ° c の孵化によって反応を不活性化します。</li>
</ol>5. リアルタイム PCR を用いた酸応答性遺伝子発現の測定
<ol><li>384 ウェル反応板の次のコンポーネントを組み合わせて: 1 μ L テンプレート cDNA、0.75 μ 10 X N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine、前方に 0.3 μ L 50 μ Mプライマー、0.3 μ L 50 μ M 逆プライマー、1 μ L の Taq のポリメラーゼ、Taq ポリメラーゼ バッファーの μ L の 20 mM dNTP、16.65 μ L 水の 2.5 x 2.5 μ L 10。 注:テーブルの材料に市販のキットの例を示します。プライマーの一覧を表 1に示します。96 ウェル リアクション プレートをこのステップで使用もできます。(例えば、Taqman プローブ) 他のプローブも使用できます。</li>
	<li>実験およびリアルタイム PCR システムに次の PCR プログラムを設定: 2 分、1 サイクルは 50 ° C95 ° C、10 分、1 サイクル95 ° C 15 s の 60 ° C 1 分、40 サイクル95 ° C、15 s、1 サイクル60 ° C で 15 秒、1 サイクル95 ° C、15 s、1 サイクル。</li>
	<li>QPCR 測定器でプレートを配置します。プログラムを起動します。 注: IDI1 INSIG1、pH 応答性遺伝子MSMO1の発現タンパク質レベルで測定できます。</li>
</ol>

<ol>
	<li>Cairns, R. A., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+cell+metabolism.">Regulation of cancer cell metabolism.</a> Nature reviews. Cancer. 11 (2), 85-95 (2011).</li><li>Chang, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Competition+in+the+Tumor+Microenvironment+Is+a+Driver+of+Cancer+Progression.">Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.</a> Cell. 162 (6), 1229-1241 (2015).</li><li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Gerweck, L. E., Seetharaman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pH+gradient+in+tumor+versus+normal+tissue:+potential+exploitation+for+the+treatment+of+cancer.">Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer.</a> Cancer research. 56 (6), 1194-1198 (1996).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nature Rev. Cancer. 11 (9), 671-677 (2011).</li><li>Gatenby, R. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+cancers+have+high+aerobic+glycolysis?.">Why do cancers have high aerobic glycolysis?.</a> Nature reviews. Cancer. 4 (11), 891-899 (2004).</li><li>McBrian, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+regulates+intracellular+pH.">Histone acetylation regulates intracellular pH.</a> Mol Cell. 49 (2), 310-321 (2013).</li><li>Damaghi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+acidosis+in+the+tumour+microenvironment+selects+for+overexpression+of+LAMP2+in+the+plasma+membrane.">Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.</a> Nat Commun. 6, 8752 (2015).</li><li>Corbet, C., 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=Acidosis+Drives+the+Reprogramming+of+Fatty+Acid+Metabolism+in+Cancer+Cells+through+Changes+in+Mitochondrial+and+Histone+Acetylation.">Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.</a> Cell Metab. 24 (2), 311-323 (2016).</li><li>Chen, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+analysis+of+lactic+acidosis+and+acidosis+response+in+human+cancers.">The genomic analysis of lactic acidosis and acidosis response in human cancers.</a> PLoS genetics. 4 (12), e1000293 (2008).</li><li>Kondo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Acidic+pH+Activates+the+Sterol+Regulatory+Element-Binding+Protein+2+to+Promote+Tumor+Progression.">Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.</a> Cell Rep. 18 (9), 2228-2242 (2017).</li></ol>

著者綾乃近藤は協和発酵キリン株式会社の従業員

ここでは、簡単な酸性 pH 文化システムとその評価プロセスについて説明しました。中程度の酸性化、すなわち、減らされた重炭酸濃度、乳酸添加と HCl 添加の 3 つの方法の組み合わせでは、pH 応答メカニズムを徹底的に調査し、他の腫瘍に対する細胞の反応を比較できました。低酸素や栄養飢餓などアイソレータケージ条件。
このメソッドのキーは、培養液中の?...

腫瘍微小環境は、腫瘍の進行と癌細胞代謝1,2,3で重要な役割を果たしています。がん細胞が低酸素血症、栄養不足、酸性細胞外 pH (pHe) など条件にさらされること。ただし、腫瘍の進行の pHe の役割は明らかにされていないとして広く低酸素や栄養飢餓のように。腫瘍組織内 pHe を酸性、およそ pH 6.84,5に到達になることができます。酸性の pH はプロトン (H+) の好気性と嫌気性解糖系の排泄から発生し、増殖癌による乳酸細胞5,6.
最近の研究で明らかに酸性 pHe 誘起ヒストン脱アセチル化、脂肪酸酸化、酸性リソソーム酸性厳しい7,8,9,10への適応のための放出。しかし、どの細胞の酸性化を通じたメカニズム、がん行動と酸性 pH 腫瘍微小環境における規制当局が完全に決定されていないキーの id。さらに、いくつかのレポートの重炭酸塩、トリス、パイプ、および HEPES バッファーまたは乳酸と HCl、不明瞭な濃度を使用して様々 な酸性 pH メディアが中も包括的な調整後の安定性を示す報告は少ないいくつかの異なる酸性培地7,8,9,10の比較
主調整装置と細胞の酸性化のコンテキストでがん細胞の代謝変化を解明するには、酸性 pHe を維持するために単純な体外培養モデルを設立しを癌細胞11pHe の役割を検討しました。37 ° C 未満 5%、6.8 の pHe と酸性培を維持してこのメソッドを使用すると、CO2培養液の減少の重炭酸濃度を使用しています。pH 7.4 は、通常どおりに使用され、媒体を制御します。培地の pH は、少なくとも 24 時間持続され、72 h PANC-1 および AsPC 1 膵臓癌細胞の培養中に徐々 に減少します。削減するのではなく、乳酸を追加することによって乳酸性アシドーシスを模倣文化方法を確立した解糖系が乳酸7,8,9では、陽子だけでなく排泄を加速するため、重炭酸塩の濃度。さらに、中で HCl による酸性 pHe は酸性 pH 培養培地への細胞は重炭酸塩の集中力の低下の原因ではない可能性を除外することが出来ます。また、6.4 〜 7.4 異なる重炭酸塩濃度の pH で様々 なメディアを使用して、我々 は、細胞応答 pHe 影響の程度を評価できます。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#8220;Nissui&#8221; 2</td>
			<td>Nissui</td>
			<td>05919</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher&#160;</td>
			<td>10438</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Thermo Fisher&#160;</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution(Stabilized)</td>
			<td>Nacalai</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5g/l-Tripsin/0.53mmol/l-EDTA Solution, with Phenol Red</td>
			<td>Nacalai</td>
			<td>32778-05</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5%-Trypan Blue Stain Solution</td>
			<td>Nacalai</td>
			<td>29853-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>252476</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>QIAGEN</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV First-Strand Synthesis System</td>
			<td>Thermo Fisher&#160;</td>
			<td>18091</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix&#160;</td>
			<td>Applied Biosystems&#160;</td>
			<td>4368702&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1</td>
			<td>ATCC</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
		<tr>
			<td>KMS-6</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB0432</td>
			<td></td>
		</tr>
		<tr>
			<td>TIG</td>
			<td>JCRB Cell Bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRC9</td>
			<td>ATCC</td>
			<td>CCL-212</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>ATCC</td>
			<td>CRL-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of an extracellular acidic ph culture system

腫瘍微小環境、低酸素や栄養飢餓などの条件は、癌の進行や悪性腫瘍で重要な役割を果たします。ただし、腫瘍の積極性とそのメカニズムの解明に酸性細胞外 pH の役割は検討されていない広く低酸素や栄養飢餓状態と比較しています。さらに、酸性細胞外腫瘍微小環境を模倣するように明確に定義された培養法は完全に報告されていません。
重炭酸塩や増加の乳酸培養液中で HCl 濃度酸性細胞外 pH による削減を維持するために単純な体外培養法をご紹介します。培地の pH は少なくとも 24 時間持続され、72 h PANC-1 および AsPC 1 膵臓癌細胞の文化を次によって徐々 に減少します。本研究では 3 つの異なる酸性メディア条件高い pH 応答性遺伝子MSMO1 INSIG1、 IDI1などと比較して低酸素や栄養飢餓.これらの遺伝子の発現は、酸性 pH のマーカーとして使用できます。これらの簡単なテクニックは、酸性腫瘍微小環境下で腫瘍の悪性度の基になるメカニズムを解明するために有益です。したがって、細胞の酸性 pH 文化体制によりがん細胞のみならず、その他酸性障害など糖尿病性ケトアシドーシスに関連して、腎の尿細管細胞など、細胞の細胞の酸性 pH 応答の発見乳酸アシドーシス、腎の尿細管性アシドーシス、呼吸性アシドーシス。

適切な重炭酸濃度を決定するには、0 - 8 mM NaHCO3 (培養液に最終濃度) の範囲で DMEM を作製し、pH 6.4 7.4 (図 1) に至ると培地を準備することに成功しました。固形腫瘍4,5pH 6.8 を細胞外 pH に達することを示す以前のレポートによると 8 mm NaHCO3 (pH 7.4) 制御媒体と 2 mM NaHCO3 (Ph6.8) 媒体と...

Watch this Scientific Journal Video about Establishment of an Extracellular Acidic pH Culture System at JoVE.com

Establishment of an Extracellular Acidic pH Culture System

酸性腫瘍微小環境は、腫瘍の進行に重要な役割を果たしています。がん細胞のin vitro での酸性細胞外 pH の効果を評価するためには、単純な酸性文化体制を確立しました。

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-of-an-extracellular-acidic-ph-culture-system

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Unit 1

Fluid, Electrolyte, and Acid-Base Balance

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14.5K ビュー.  Kyowa Hakko Kirin Co., Ltd. 酸性腫瘍微小環境は、腫瘍の進行に重要な役割を果たしています。がん細胞のin vitro での酸性細胞外 pH の効果を評価するためには、単純な酸性文化体制を確立しました。

ビデオ: Establishment of an Extracellular Acidic pH Culture System

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

酸性pHでとα-エクスパンにより誘起される植物細胞壁の延長（クリープ）を測定

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

生物学

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

アン EX - OVO</emイメージングとマイクロサーアプリケーションに適して>ニワトリ胚培養システム

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

腱板断型ボトルの培養システムを使用してげっ歯類全胚培養の方法

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

アン<em&gt;生体外</em甲状腺の開発を研究する&gt;文化システム

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

細胞間接触の不在で2つの細胞タイプの挿入共培養システムの開発

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

蛍光遺伝子にコードされた pH インジケーターとキイロショウジョウバエのマルピギー氏管の上皮細胞で光の定量化の細胞内 pH

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

抱合の単細胞藻類の栄養文化の確立

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

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

自動イメージングシステムを用いたiPS細胞の合流点の測定

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

凍結保存された上皮陰窩からの子豚腸3Dオルガノイドの培養と細胞単分子膜の樹立

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

微生物叢マルチオミクスのための細菌細胞外小胞の単離の強化

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

著者スポットライト:細菌の細胞外小胞の分離と不均一性に関する研究の加速 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.