我々 は特にマーク ・ ヘンソン、ジェレミー ・ グラハムとジョアン ・ コレイア、このシステムの開発に協力してくださった、ケアン研究を感謝したいです。我々 もありがとうデイヴィッド ウェールズ語とアクヒレシュ ・ レディ (旧浜松市) のピーター ・ ラスキーと同様、設計段階の間に貴重な議論のための原稿に彼の重要な入力デモ カメラとデビッド ・ ウォンのローンの手配します。

<ol>
	<li>Morgan, L. W., Greene, A. V., Bell-Pedersen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+and+light-induced+expression+of+luciferase+in+Neurospora+crassa.">Circadian and light-induced expression of luciferase in Neurospora crassa.</a> Fungal Genet. and Biol. 38 (3), 327-332 (2003).</li><li>Millar, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Circadian+Phenotype+Based+on+Firefly+Luciferase+Expression+in+Transgenic+Plants.">A Novel Circadian Phenotype Based on Firefly Luciferase Expression in Transgenic Plants.</a> Plant Cell Online. 4 (9), 1075-1087 (1992).</li><li>Yu, W., Hardin, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Firefly+Luciferase+Activity+Assays+to+Monitor+Circadian+Molecular+Rhythms+In+Vivo+and+In+Vitro.">Use of Firefly Luciferase Activity Assays to Monitor Circadian Molecular Rhythms In Vivo and In Vitro.</a> Circadian Rhythms: Methods and Protocols. , 465-480 (2007).</li><li>Yoo, S. -. 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=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc Natl Acad Sci. 101 (15), 5339-5346 (2004).</li><li>Buhr, D. E., Yoo, S. -. H., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+as+a+universal+resetting+cue+for+mammalian+circadian+oscillators.">Temperature as a universal resetting cue for mammalian circadian oscillators.</a> Science. 330 (6002), 379-385 (2010).</li><li>Brown, S. A., Zumbrunn, G., Fleury-Olela, F., Preitner, N., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythms+of+mammalian+body+temperature+can+sustain+peripheral+circadian+clocks.">Rhythms of mammalian body temperature can sustain peripheral circadian clocks.</a> Current Biology. 12 (18), 1574-1583 (2002).</li><li>Balsalobre, 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=Resetting+of+circadian+time+in+peripheral+tissues+by+glucocorticoid+signaling.">Resetting of circadian time in peripheral tissues by glucocorticoid signaling.</a> Science. 289 (5488), 2344-2347 (2000).</li><li>Balsalobre, A., Marcacci, L., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+signaling+pathways+elicit+circadian+gene+expression+in+cultured+Rat-1+fibroblasts.">Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts.</a> Curr. Biol. CB. 10 (20), 1291-1294 (2000).</li><li>Hastings, M. H., Reddy, A. B., McMahon, D. G., Maywood, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circadian+mechanisms+in+the+suprachiasmatic+nucleus+by+transgenesis+and+biolistic+transfection.">Analysis of circadian mechanisms in the suprachiasmatic nucleus by transgenesis and biolistic transfection.</a> Methods Enzymol. 393, 579-592 (2005).</li><li>Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Ba Barres, ., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NS21:+re-defined+and+modified+supplement+B27+for+neuronal+cultures.">NS21: re-defined and modified supplement B27 for neuronal cultures.</a> J. Neurosci Methods. 171 (2), 239-247 (2008).</li><li>Feeney, K. A., Putker, M., Brancaccio, M., ONeill, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+Characterization+of+Firefly+Luciferase+as+a+Reporter+of+Circadian+Gene+Expression+in+Mammalian+Cells.">In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.</a> J. Biol. Rhythms. 31 (6), 540-550 (2016).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Hirota, T., Lewis, W. G., Liu, A. C., Lee, J. W., Schultz, P. G., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+biology+approach+reveals+period+shortening+of+the+mammalian+circadian+clock+by+specific+inhibition+of+GSK-3beta.">A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta.</a> Proc Natl Acad Sci. 105 (52), 20746-20751 (2008).</li><li>Meng, Q., Maywood, E. S., Bechtold, D. A., Lu, W., Li, J., Gibbs, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrainment+of+disrupted+circadian+behavior+through+inhibition+of+casein+kinase+1+(+CK1+)+enzymes.">Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 ( CK1 ) enzymes.</a> Proc Natl Acad Sci. 1, 1-6 (2010).</li><li>Khabirova, E., Chen, K. -. F., O'Neill, J. S., Crowther, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flyglow:+Single-fly+observations+of+simultaneous+molecular+and+behavioural+circadian+oscillations+in+controls+and+an+Alzheimer's+model.">Flyglow: Single-fly observations of simultaneous molecular and behavioural circadian oscillations in controls and an Alzheimer's model.</a> Sci. Rep. 6, 33759 (2016).</li></ol>

利益相反は存在しません。

ここで説明されているプロトコルは、哺乳類細胞培養、灌流および静的な条件の下で両方のです。しかし、ワニは簡単に他のモデル システムに適応できます。確かに、それが運動、睡眠とキイロショウジョウバエ暗黒15.の下で維持される周辺遺伝子発現リズムの同時監視するための優れたプラットフォームを提供するために示されて既にいます。ここに記載されている以外のカメラの種類のアプリケーションによっては、適切にする可能性がありますをまた指摘しました。我々 は、適切なフィルターで変更されたバージョンの現在のセットアップされる可能性がありますプリンシパルの蛍光定量を想定します。
ワニできない可能性があります適切な唯一のアプリケーションは、特に高空間分解能は、哺乳類の脊髄スライスにおける PER2::LUC 発現の時空間組織のイメージングなど、必要です視交叉上核やその他の小さな切片。
ワニは、これまでの従来の録音技術によって容易に達成されていない実行する多くの実験できます。生物発光の測定に対する現在の方法と比べると、ワニは、細胞培養ディッシュまたは使用できるスライド、外部メディア条件、感度、およびドメインの両方のタイプの柔軟性の向上を提供します。
これは特に関連して同時に 3 D organoid 文化システムに向けて標準的な 2次元細胞モデルから移動がある場合。など、ワニが多くの幾日および週の条件の広い範囲の下に、生物発光を測定できます適応メソッドを提供すると予想されます。

ルシフェラーゼ遺伝子の発現と蛋白質の活動の記者としての分子細胞生物学研究の一般的な手法となっています。これはホタルのルシフェラーゼの合成と触媒不活性化の速度が約 24 h の概日サイクルにわたって発生する遺伝子発現の変化をレポートに特に適して概において当てはまります。など、ルシフェラーゼは有機体、菌類、植物、ハエ、哺乳類1,2,3、4などの広い範囲にわたって概記者として採用されています。
概日遺伝子発現の in vitroの定量化、光電子増倍管 (PMT) は発光信号を記録する通常使用されます。光電子増倍管を用いた測定柔軟性が限られてしかし、通常、あらかじめ決められた版か皿サイズに制限されています。じゃはまたイメージングのルシフェラーゼ発現のばらつきを示すサンプル情報の損失につながることができる光電子増倍管を使用して監視のサンプルから空間情報を収集することが可能ではないです。また、光電子増倍管と関連する電子工学標準細胞文化のインキュベーターの加湿環境にさらされたときに誤動作しやすいが、光電子増倍管を用いた縦ルシフェラーゼ記録は非加湿インキュベーターで常に実行されます。その結果、細胞培養皿の気密性の高い蒸発による水分の損失を防ぐために密封されなければならないし、文化メディアは 3-したがってバッファーする必要があります (Nmorpholino) propanesulfonic 酸 (モップ) または 4-(2-ヒドロキシエチル)-1-piperazineethanesulfonic 酸 (HEPES) ではなく、生体内で機能し、哺乳類の組織培養で日常的に使用されているシステムをバッファリング CO2/bicarbonate。
これらの制限の結果として光電子増倍管による生物発光の測定は通常細胞、実験間は保持される条件に制約を配置します。我々 が、標準的な CO2/N2 170 L 培養インキュベーター、水・低温電子-乗算の添加により適応したを使用してこれらの問題を克服するために、また可能な実験条件の範囲を拡大するには曇らない光学およびデジタル制御温度やガスの電荷結合素子 (EMCCD) カメラ。これは縦ルシフェラーゼ イメージング ガスの自動化や Temperature-Optimized レコーダー、ワニと呼ばれています。ワニ生物発光イメージング、両方標準的な組織培養プレートの高速イメージングのための大幅に高められた柔軟性を可能 (最大 6 x 96 または 384-ウェル プレート同時に) およびまた標準培養システム、そのような灌流セルとしてマイクロ流体デバイスで栽培。この楽器も両方 CO2 O2分圧と温度可変制御と加湿条件下で発生するイメージングできます。
以下のプロトコルでは、哺乳類の細胞の培養システム (今後 '発光インキュベーター' というワニを使用して発光記録する方法について説明します。しかし、システム生物発光イメージング、また、いくつかの変更は、他の生物学的システムとコンテキストの数でのイメージングに適してこと、注意すべきであります。

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM (1x) + GlutaMAX</td>
			<td style="width:145px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hyclone FetalClone III Serum</td>
			<td style="width:145px;">GE Healthcare</td>
			<td>SH30109.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
			<td style="width:145px;">Thermofisher</td>
			<td>21103049</td>
			<td>basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:145px;">Sigma</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Catalase</td>
			<td style="width:145px;">Sigma</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutathione</td>
			<td style="width:145px;">Sigma</td>
			<td>G6013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:145px;">Sigma</td>
			<td>I1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superoxide Dismutase</td>
			<td style="width:145px;">Sigma</td>
			<td>S5395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Holo-transferrin</td>
			<td style="width:145px;">Calbiochem</td>
			<td>616424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T3 (triiodo-L-thyronine</td>
			<td style="width:145px;">Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Carnitine</td>
			<td style="width:145px;">Sigma</td>
			<td>C7518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanolamine</td>
			<td style="width:145px;">Sigma</td>
			<td>E9508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D (+)-Galactose</td>
			<td style="width:145px;">Sigma</td>
			<td>G0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Putrescine</td>
			<td style="width:145px;">Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Selenite</td>
			<td style="width:145px;">Sigma</td>
			<td>S9133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Corticosterone</td>
			<td style="width:145px;">Sigma</td>
			<td>C2505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linoleic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>L1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linolenic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>L2376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipoic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>T1395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Progesterone</td>
			<td style="width:145px;">Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retinol Acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>R7882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retinol, all trans</td>
			<td style="width:145px;">Sigma</td>
			<td>95144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D,L-alpha-Tocopherol</td>
			<td style="width:145px;">Sigma</td>
			<td>95240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D,L-alpha-Tocopherol acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>T3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate Solution</td>
			<td style="width:145px;">Sigma</td>
			<td>S8761-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GlutaMAX (100x)</td>
			<td style="width:145px;">Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:145px;">Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galaxy 170R incubator&#160;</td>
			<td style="width:145px;">Eppendorf</td>
			<td style="width:119px;">CO17301001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin</td>
			<td style="width:145px;">Biosynth</td>
			<td style="width:119px;">L-8220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D -(+)-Glucose solution</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">G8644-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">D5030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MOPS</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">PHG0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mm I.D. silicone tubing&#160;</td>
			<td style="width:145px;">GE Healthcare</td>
			<td>19-4692-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Elbow luer connector</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Male luer fittings</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Female luer fittings</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#181;-slide luer I 0.6</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">80196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD plastipak 20ml syringe</td>
			<td style="width:145px;">Becton Dickinson</td>
			<td style="width:119px;">300613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1mm I.D. ETFE tubing</td>
			<td style="width:145px;">GE Healthcare</td>
			<td style="width:119px;">18-1142-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PF670462</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">SML0795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B27 Supplement (50x)</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iXon Ultra EMCCD camera</td>
			<td style="width:145px;">Andor</td>
			<td style="width:119px;">iXon 888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiji</td>
			<td style="width:145px;">ImageJ</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Prism 7.0</td>
			<td style="width:145px;">Graphpad Software</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deltaphase Isothermal Pad</td>
			<td style="width:145px;">Braintree Scientific</td>
			<td style="width:119px;">39DP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated neutral density filter&#160;</td>
			<td style="width:145px;">Cairn Research</td>
			<td style="width:119px;">Custom item</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmomat 030</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">Discontinued</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">300 mOsmol/kg calibration standard</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">30.9.0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Measuring vessel</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">30.9.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Focusing cylinder</td>
			<td style="width:145px;">Cairn Research</td>
			<td style="width:119px;">Custom item</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NE-1600 programmable syringe pump</td>
			<td style="width:145px;">Pump Systems inc.</td>
			<td style="width:119px;">NE-1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor Solis Software</td>
			<td style="width:145px;">Andor</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

flexible measurement of bioluminescent reporters using an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (alligator)

細胞の遺伝子発現のレポーターをルシフェラーゼ ベースは、縦方向の両方の広範な使用とエンドポイントを生物学的活性の試金。概日リズム研究のたとえば、ホタルのルシフェラーゼと時計遺伝子の融合を生じさせる堅牢なリズムで数日間保持細胞生物発光。技術的な制限は、光電子増倍管 (PMT) に関連付けられているまたは発光定量化のため従来の顕微鏡ベースの方法は通常の間にかなり非生理的条件下で細胞や組織が維持されることを要求しています。感度とスループットのトレードオフで録音。ここで、異なる高感度可変ガス ・湿度制御を含む培養条件の広い範囲をサポートする、受け入れる多くのスループットと長期的な生物発光イメージングを可能にする従来の方法の改良を報告します。組織培養プレートと料理。またガスと温度最適化レコーダー (ワニ) をイメージングこの自動縦ルシフェラーゼ単層細胞またはティッシュ、従来容易に観察することはできません間ルシフェラーゼ発現の空間変動の観測が可能します。メソッド。我々 はワニがルシフェラーゼ活性既存手法と比較した場合の検出の非常に高い柔軟性を実現する方法を強調表示します。

この記事では、生物発光イメージング ワニ (発光インキュベーター) を使用して哺乳類セルのためのプロトコルについて説明します。この手法により物理的なセットアップおよび細胞外の条件の柔軟生物発光システムをイメージングするとき。単純な静的培養 (図 1、補助ビデオ 1) と灌流培養 (図 2、附則のビデオ 2) の両方の方法を説明しますがこのシステムを使用して他の多くのセットアップを描出できた。すべてのデータは、セクション 7 に記載されている方法を使用して定量化されました。
図 1 aは、生物発光 PER2::LUC 線維芽細胞4を含む 6 x 96 ウェルのプレートからの録音のビデオの例を示します。最も外側の井戸は、細胞を含まない、これらがこの実験のために必要ではなかった。セル録音のため一定の 37 ° C で開催されている前に後 12 h 72 h または (12 h、12 h、72 h 32 ° C で続いて 37 ° c)、コンバースの 37 ° C に 32 ° c、12 時間のいずれかの温度サイクルを受けるという温度差同調してきました。 60 分エクスポー ジャーは、すべての時間を撮影されました。これらの条件の 2 つが図 1 bで定量化しました。
図 2 aは、灌流培養システムのセットアップの概略を示しています。これは、チューブで接続されている 2 つのチャネルのスライドで構成されています。メディアは、シリンジ ポンプによってセルを介して駆動されます。これらのスライドの最初のガス透過性バブル トラップ (バッファー スライド) として機能し、2 番目のセルが含まれていない生物発光の記録から細胞を含みます。このシステムからビデオを録画代表を図 2 bに示します。15 分のエクスポー ジャーは、15 分毎に撮影されました。標準灌流条件下またはカゼインのキナーゼ阻害剤を概日周期を長くして、培養における時計遺伝子発現リズムの振幅を低減以前示されている PF670462 の存在下での細胞の維持、ここで哺乳類セル14。PER2::LUC 発現に及ぼす影響は期間の定量化 (底板)、図 2に示す標準的な静的な細胞培養条件下での薬物の濃度が同じ細胞に対して図 2 (最上位のパネル) に示すように図 2 Dで表示されます。PF670462 による治療条件の両方のセットの下で PER2::LUC 式に影響を与えることこれから明らかです。ただし、灌流条件のショー期間延長約 3 h (3 発熱量 h) の下で PF670462 で処理した細胞、間薬剤と扱われる静的な条件のセル表示の期間により大幅に期間延長 &#62; 48 h。これは、減衰のコサインが第 7 節で説明するように適合 (余分な二乗和 F テスト対直線 p &#60; 0.0001)。興味深いことに、灌流の下で期間延長の大きさは、生体内で14を観察にかなり近いです。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56623/56623fig1v2.jpg"> 
図 1: データの例です。6 x 96 で不死化 PER2::LUC の生物発光のビデオ スナップ ショットはよく発光インキュベーターのプレート (A) の例。定量化する井戸は、附則のビデオ 1で黄色で強調されています。(B) A からの発光の定量化逆 PER2 タンパク質発現相を生成する互いに反一過性だった温度サイクル (12 h は、32 ° c; 12 h、37 ° C で) を使用してセルの 3 プレートの 2 セットが噴流床 (n = 6, ± SEM を意味する)。<a href="//ecsource.jove.com/files/ftp_upload/56623/56623fig1v2large.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/56623/56623fig2v2.jpg"> 
図 2: CK1δ 阻害剤による血液灌流します。(A) 灌流システムの模式図。(B) 例 PER2::LUC から記録生物発光のビデオ スナップ ショットの灌流下のセルします。定量化のサンプル領域が黄色で強調表示されます。(C) PER2::LUC 線維芽細胞灌流の下で、3 μ M CK1 阻害剤 PF670462 と静的条件下での発光の定量化 (n = 3、± SEM を意味する)。生物発光は、最小値と最大値に正規化されています。期間 (双方向分散分析, ホルム Sidak の多重比較検定) の (D) 分析。<a href="//ecsource.jove.com/files/ftp_upload/56623/56623fig2v2large.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>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>コンポーネント</td>
			<td>最終的な中濃度 (μ M)</td>
			<td>株式 (mg/mL)</td>
			<td>400 mL NS21 の</td>
		</tr>
		<tr>
			<td>牛のアルブミン</td>
			<td>37</td>
			<td>-</td>
			<td>50 g</td>
		</tr>
		<tr>
			<td>カタラーゼ</td>
			<td>0.01</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>グルタチオン</td>
			<td>3.2</td>
			<td>-</td>
			<td>20 mg</td>
		</tr>
		<tr>
			<td>インスリン</td>
			<td>0.6</td>
			<td>10</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>スーパーオキシドジスムターゼ</td>
			<td>0.077</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>ホロ トランスフェリン</td>
			<td>0.062</td>
			<td>-</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>T3 (triiodo L サイロニン)</td>
			<td>0.0026</td>
			<td>2</td>
			<td>20 Μ L</td>
		</tr>
		<tr>
			<td>L カルニチン</td>
			<td>12</td>
			<td>-</td>
			<td>40 mg</td>
		</tr>
		<tr>
			<td>エタノールアミン</td>
			<td>16</td>
			<td>液体 (1 g/ml)</td>
			<td>20 Μ L</td>
		</tr>
		<tr>
			<td>D-(+)-ガラクトース</td>
			<td>83</td>
			<td>-</td>
			<td>300 mg</td>
		</tr>
		<tr>
			<td>プトレシン</td>
			<td>183</td>
			<td>-</td>
			<td>322 mg</td>
		</tr>
		<tr>
			<td>亜セレン酸ナトリウム</td>
			<td>0.083</td>
			<td>1</td>
			<td>280 Μ</td>
		</tr>
		<tr>
			<td>コルチコステロン</td>
			<td>0.058</td>
			<td>2</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>リノール酸</td>
			<td>3.5</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>リノレン酸</td>
			<td>3.5</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>リポ酸</td>
			<td>0.2</td>
			<td>4.7</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>プロゲステロン</td>
			<td>0.02</td>
			<td>3.2</td>
			<td>0.04 mL</td>
		</tr>
		<tr>
			<td>レチノール アセテート</td>
			<td>0.2</td>
			<td>20</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>レチノール、すべてトランス</td>
			<td>0.3</td>
			<td>10</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>D、L-α-トコフェ ロール</td>
			<td>2.3</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>D、L-α-トコフェ ロール酢酸</td>
			<td>2.1</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
	</tbody>
</table>
表 1: NS21 準備。
補助ビデオ 1: ウェル プレートの生物発光例ビデオ。生物発光のインキュベーターで 6 x 96 ウェル プレートで不死化 PER2::LUC の生物発光例ビデオ スナップ ショット。定量化する井戸は、黄色で強調表示されます。<a href="http://ecsource.jove.com/files/ftp_upload/56623/Video_1A_Revision-1.avi">このファイルをダウンロードするここをクリックしてください</a>。
補助ビデオ 2: PER2::LUC から記録生物発光の例ビデオ スナップ ショットの灌流下のセルします。定量化のサンプル領域が黄色で強調表示されます。<a href="http://ecsource.jove.com/files/ftp_upload/56623/Video_2B_Revision-1.avi">このファイルをダウンロードするここをクリックしてください</a>。

Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

遺伝的コード化ルシフェラーゼは、遺伝子発現の人気のある非侵襲的なレポーターです。ガスと温度最適化レコーダー (ワニ) をイメージング自動縦ルシフェラーゼを使用できます、条件の広い範囲の下で発光細胞から長手記録です。ワニ、概日リズム研究のコンテキストで使用される方法を紹介します。

ガスと温度最適化レコーダー (ワニ) をイメージング自動縦ルシフェラーゼを用いた発光レポーターの柔軟な測定

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

flexible-measurement-bioluminescent-reporters-using-an-automated

Research

JoVE Journal

Biology

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

7.8K ビュー.  MRC Laboratory of Molecular Biology. 遺伝的コード化ルシフェラーゼは、遺伝子発現の人気のある非侵襲的なレポーターです。ガスと温度最適化レコーダー (ワニ) をイメージング自動縦ルシフェラーゼを使用できます、条件の広い範囲の下で発光細胞から長手記録です。ワニ、概日リズム研究のコンテキストで使用される方法を紹介します。

ビデオ: Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

IVISスペクトラムを用いた乳腺腫瘍の in vivo生物発光イメージングに

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

生物学

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

ゼブラフィッシュの化学スクリーンのための自動化された画像と解析の開発。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

ルシフェラーゼ生物発光レポーターを用いた遺伝子発現のモニタリング細胞自律的な概日時計のリズム

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)7. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices8. To date, leaf shape programs (e.g. SHAPE9, LAMINA10, LeafAnalyzer11, LEAFPROCESSOR12) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size13. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.

LeafJ：半自動化された葉の形状計測のためのImageJのプラグイン

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

ゼブラフィッシュ胚における組織特異的蛍光の半自動イメージング

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

イオン選択自己参照微小電極法を用いた細胞外イオンフラックスの測定

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

ショウジョウバエの調製と光コヒーレンス顕微鏡を用いてin vivoで心機能の縦イメージング（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

3Dスフェロイド生産のための効率的で柔軟な細胞凝集法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

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 ...