Name: a microfluidic platform for longitudinal imaging in caenorhabditis elegans
Uploaded: 2018-05-02T13:30:00.000+00:00
Duration: 9 min
Description: Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

この研究は、PHY 1205494 を通じて全米科学財団および MCB 1413134 (EL)、韓国の国立研究財団助成金 2017R1D1A1B03035671 にサポートされていた (KSL)。

以下のプロトコルは、WormSpa5ワームの縦画像の説明したマイクロ流体デバイスを使用します。(リクエストの作成者から入手できる CAD ファイルから始まる) WormSpa の作製は簡単ですが、いくつかの専門知識が必要です。ほとんどの場合、中核施設やそのようなサービスを提供する商業企業は、作製を行う容易にことが。機能の高さは 50 μ m であることを指定するのには、デバイスを製造するときを確認します。
1. 実験装置
<ol><li>電動ステージと倒立蛍光顕微鏡のマイクロ流体デバイスをマウントします。</li>
	<li>顕微鏡に近い軌道シェーカーを配置、シェーカーから振動顕微鏡に直接影響しないかどうかを確認します。たとえば、光学テーブルとの接触を一切壁に固定棚にシェーカーを置きます。</li>
	<li>軌道のシェーカーにシリンジ ポンプを配置します。それは揺れながらプルプルしないので、シェーカーにポンプをテープします。</li>
</ol>2. マイクロ流体環境の準備
注: 実験中に 4 つの注射器に接続している注射器のチューブを介してマイクロ流体デバイス図 1で説明したよう。この手順では、これらの注射器の準備について説明します。特に記述がない限り、タウトをせずできるだけ短く注射器チューブを維持します。
<ol><li>アウトレットの注射器と注射器チューブを準備します。この注射器 (図 1では、S1) は後で注射器の管を通って装置のコンセントに接続され、デバイスから出てくる流体の一時ストレージに使用します。<ol>
<li>アダプター針を針 (金属部分) のみを維持する 20 x 1/2 インチのシリンジの先端のプラスチックの部品を削除してください。プラスチックや金属パーツをペンチで押しそれらを引き離すようにします。針の残りのプラスチック残渣は、ブンゼン バーナーと焼くことができます。</li>
		<li>ペンチでその 2 つの端を押しながら実験のセットアップの幾何学によると針を曲げます。ここで説明した設計、その真ん中に針を 〜 110 ° の角度に曲げ、最も便利です。</li>
		<li>アダプター針半分の方法をチューブに差し込みます。他の半分は後でデバイスに接続されます。注射器のチューブ リーク (たとえば、0.86 mm の内径と外径 1.32 mm チューブ) なし 20 G シリンジの先端と互換性があります。推奨チューブの長さは、~ 50 cm です。</li>
		<li>10 mL ルアーロック シリンジをシリンジ チューブ 20 x 1/2 インチ シリンジ チップを介しての他の (開いている) 端に接続します。</li>
	</ol>
</li>
	<li>バッファー入口注射器を準備し、チューブを注射器します。これらの注射器の 1 つ (図 1の S2) が行くデバイスおよび注入の流れを制御する流体の貯水池として使用されます。その他の注射器 (図 1の S3) はバッファー交換に必要な手順 3.2 で説明したよう。<ol>
<li>注射器チューブ (図 1) を介して同じ弁三方弁に直接 10 mL ルアーロック シリンジ (S2) を接続し、別の注射器 (S3): シリンジ チップを介してチューブに S3 を接続し、バルブを介して別のシリンジの先端にチューブを接続します。 注: これらの材料は、接続されている順序は関係ありません。</li>
		<li>三方弁の残りのポートに注射器のチューブを接続します。ステップ 2.1.2 のように別のアダプターの針を準備、注射器管のもう一方の (オープン) の端に針を半分の方法を差し込みます。</li>
		<li>S3 が閉じている間、2.2.2 のステップと S2 で注射器管が接続されているようなバルブを設定 (図 1)。</li>
	</ol>
</li>
	<li>ワーム入口の注射器と注射器チューブを準備します。この注射器 (図 1の S4) がデバイスにワームを読み込むために使用されます。<ol>
<li>10 mL ルアーロック シリンジ (S4) に長い注射器のチューブを接続します。読み込み用に使用する流体の量からこのチューブの長さを決定します。たとえば、100 cm のチューブの液体 2 mL 以上をサポート (また、手順 4.2 を参照)。</li>
		<li>2.1.2 の手順のように別のアダプターの針を準備し、片方注射器管の他の (開いている) 最後に針の半分。</li>
	</ol>
</li>
	<li>すべての注射器とチューブ S 中12回すすぎます。S 媒体、すべての空気の泡を削除に世話、注射器やチューブを入力します。</li>
	<li>装置のコンセントにコンセント注射器のチューブを接続します。<ol>
<li>出口ポートに注射器管端アダプター針を差し込みます。</li>
		<li>少し S 中はバッファー入口とワームの入口から出てくるまでにデバイスを埋めるためコンセントを通じて S 媒体の少量を注入します。</li>
	</ol>
</li>
	<li>ピン (1/32 インチ径ステンレス鋼ダウエルピン) ワーム入口を接続しながら、デバイスのバッファー入口ポートにバッファー入口注射器チューブを接続します。</li>
	<li>デバイスにバッファーの一定の流れを挿入します。シリンジ ポンプ13、上にバッファー入口シリンジ S2 を読み込み、S2 内の媒体は 3 μ L/分の流量でデバイスに連続的に注入するようにポンプを設定します。</li>
</ol>3. マイクロ流体デバイスへの細菌の読み込み
<ol><li>エシェリヒア属大腸菌OP50 S 中12で中断を準備します。<ol>
<li>標準プロトコル LB と単一コロニーの 50 mL の 250 mL フラスコに接種し、37 ° C で一晩インキュベート</li>
		<li>10 分間 4000 rpm で一晩かけて培養を遠心し、S 培地 30 mL にペレットを再懸濁します。</li>
		<li>5 μ m のシリンジ フィルターを介して懸濁液をフィルターします。600 でその光の密度によって細菌濃度を測定 nm (外径600) 外径 φ600 3 濃度を調整。この高密度は、ワームの供給を維持する必要があります。</li>
	</ol>
</li>
	<li>OP50 サスペンション デバイスのバッファーを交換します。<ol>
<li>OP50 懸濁液で満たされた清潔なシリンジ (S5) を準備します。注射器を垂直に保持し、それの上部にすべての気泡を収集する数回をタップします。</li>
		<li>デバイスへの接続を閉じますバッファー入口注射器の三方弁を切り、注射器 2 S2 と S3 を接続します。シリンジ ポンプから S2 を外します。</li>
		<li>バルブから S2 を外し、バルブに S5 を接続します。バルブを介しての S3 に S5 の上部に収集し、その少し OP50 バッファーの S3 に入るまでのすべての空気を押し出します。そう、S5 またはバルブで空気の泡が残っていないことを確認します。</li>
		<li>S3 と S5 の間の接続を閉じます、デバイスに S5 を接続し元の位置に戻る 3 方向バルブを回します。シリンジ ポンプに S5 をロードします。ポンプを保持する軌道のシェーカーを入れます。揺れの速度を約 200 rpm に設定します。</li>
		<li>100 μ L/分の流量を設定します。デバイスでワームに到着する新しいバッファーにかかる時間は、バッファー入口注射器チューブ (上記チューブで約 5 分) の長さに依存します。高速バッファー交換が必要な場合、高い流量を用いることができます。</li>
		<li>OP50 バッファーはデバイスに広がる、かつては、流量を設定 3 μ L/分に戻します。</li>
	</ol>
</li>
</ol>4. マイクロ流体デバイスにワームを読み込む
<ol><li>小さな低結合遠心チューブ (650 μ L) を準備し、OP50 懸濁液 100 μ L でそれを埋めます。まわりの伝達の 20-30 歳同期若い成虫 (46 時間後 25 ° C での L1 幼虫逮捕) NGM (線虫成長媒体) 寒天からチューブにワームで一つずつそれらを選ぶことによって選ぶ。次の標準プロトコル12歳同期を行うことができます。</li>
	<li>ワーム入口を埋める OP50 懸濁液管 (図 1の S4) をシリンジと注射器。ワームと流体の 〜 500 μ L の遠心管に挿入します。ワーム入口注射器管にワームを懸濁液を描画します。注射器管が十分な長さを確認 (> 1 m) S4 注射器に描かれたワームが注射器管に滞在し、そうでないとします。</li>
	<li>S4 をワーム インレットに接続します。マイクロ流体デバイスにすべてのワームはワーム入口注射器管を挿入します。S4 と注射器チューブを外します。ピンでワーム入口を差し込みます。</li>
	<li>機械ポンプ (図 1) からバッファー入口シリンジ S2 を外し、別のチャネルにすべてのワームをプッシュするには、S1 と S2 を手動で制御します。S2 を押すことにより、S1 を押すと逆方向にそれらを移動が、そのチャンネルにワームを移動します。チャネルが読み込まれると、各チャネルでワームは他のワームのエントリをブロックします。</li>
	<li>ワームの新しい環境に 2-3 時間の調整をしましょう。</li>
</ol>5. イメージング プロトコルを設定
<ol><li>安全にデバイスにあるすべてのワームを見つけるし、制御あなたの顕微鏡画像集録ソフトウェアでそれらのそれぞれの画像の位置を設定します。いくつかのケースで (例えば高倍率が必要な場合、または小さい CCD センサーのカメラを使用して) 複数の画像の位置が各チャンネルに必要。 注: これは手動で行うことができます、いくつかのソフトウェア パッケージは画像の撮影場所の位置を一覧表示するテキスト ファイルを読み込むことができます。これらの位置は WormSpa で定期的に並べられて、ユーザーはそのようなリストを自動的に作成する (例えばPython や商用ソフトウェア) の小さなスクリプトを書くことができます。このスクリプトの正確な形式は、取得ソフトウェア (補足資料 1の例を参照) されたファイル形式によって異なります。</li>
	<li>タイムラプス観察の必要な周波数を設定します。たとえば、感染症への免疫の遺伝子応答のイメージングは、フレームあたり 10 分の頻度で行われます。すべての時点ですべて必要なチャンネル (例:明視野、GFP 蛍光) を取得ことを確認します。</li>
</ol>6. ホストに対して緑膿菌感染症
注: この手順は、特定ホスト病原体の相互作用を研究するためです。また、1 つは興味 (生物的・非生物的ストレス、薬、シグナリング分子、等) の他の環境の手がかりが含まれているバッファーを準備できます。
<ol><li>緑膿菌PA14 SK 中14で中断を準備します。<ol>
<li>50 ml の LB と単一コロニーの 250 mL フラスコに接種し、37 ° C で一晩インキュベート</li>
		<li>50 mL の LB のと、文化の別の 250 mL フラスコに接種し、37 ° C で一晩インキュベート</li>
		<li>10 分間 4000 rpm で一晩かけて培養を遠心し、SK 培地 10 mL にペレットを再懸濁します。細菌の濃度を測定し、OD600に濃度を調整 = 4。</li>
		<li>きれいなフラスコに懸濁液を転送し、37 ° C 24 時間と揺れながら 24 時間は、25 ° C で孵化させなさい。この手順は、アッセイの14を殺す標準平板培養ステップを模倣します。</li>
		<li>5 μ m のシリンジ フィルターを介して懸濁液をフィルターします。これはデバイスを目詰まりから細菌の凝集を防ぐためです。</li>
	</ol>
</li>
	<li>ステップ 3.2 のように同じ手順に従って PA14 サスペンション デバイスで OP50 サスペンションを交換してください。10 時間のイメージングを維持します。</li>
</ol>

<ol>
	<li>Lopez-Maury, L., Marguerat, S., Bahler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+gene+expression+to+changing+environments:+from+rapid+responses+to+evolutionary+adaptation.">Tuning gene expression to changing environments: from rapid responses to evolutionary adaptation.</a> Nat Rev Genet. 9 (8), 583-593 (2008).</li><li>de Nadal, E., Ammerer, G., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+gene+expression+in+response+to+stress.">Controlling gene expression in response to stress.</a> Nat Rev Genet. 12 (12), 833-845 (2011).</li><li>Hulme, S. E., Shevkoplyas, S. S., Samuel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+Streamlining+discovery+in+worm+biology.">Microfluidics: Streamlining discovery in worm biology.</a> Nat Methods. 5 (7), 589-590 (2008).</li><li>San-Miguel, A., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+as+a+tool+for+C.+elegans+research.">Microfluidics as a tool for C. elegans research.</a> WormBook. , (2013).</li><li>Kopito, R. B., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durable+spatiotemporal+surveillance+of+Caenorhabditis+elegans+response+to+environmental+cues.">Durable spatiotemporal surveillance of Caenorhabditis elegans response to environmental cues.</a> Lab Chip. 14 (4), 764-770 (2014).</li><li>Mishra, B., 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=Using+microfluidics+chips+for+live+imaging+and+study+of+injury+Responses+in+Drosophila+larvae.">Using microfluidics chips for live imaging and study of injury Responses in Drosophila larvae.</a> J Vis Exp. (84), e50998 (2014).</li><li>Grisi, 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=NMR+spectroscopy+of+single+sub-nL+ova+with+inductive+ultra-compact+single-chip+probes.">NMR spectroscopy of single sub-nL ova with inductive ultra-compact single-chip probes.</a> Sci Rep. 7, 44670 (2017).</li><li>Crane, M. M., Chung, K., Stirman, J., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-enabled+phenotyping,+imaging,+and+screening+of+multicellular+organisms.">Microfluidics-enabled phenotyping, imaging, and screening of multicellular organisms.</a> Lab Chip. 10 (12), 1509-1517 (2010).</li><li>Leifer, A. M., Fang-Yen, C., Gershow, M., Alkema, M. J., Samuel, A. D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+manipulation+of+neural+activity+in+freely+moving+Caenorhabditis+elegans.">Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans.</a> Nat Meth. 8 (2), 147-152 (2011).</li><li>Lee, K. S., Lee, L. E., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HandKAchip+-+Hands-free+killing+assay+on+a+chip.">HandKAchip - Hands-free killing assay on a chip.</a> Sci Rep. 6, 35862 (2016).</li><li>Lee, K. S., 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=Serotonin-dependent+kinetics+of+feeding+bursts+underlie+a+graded+response+to+food+availability+in+C.+elegans.">Serotonin-dependent kinetics of feeding bursts underlie a graded response to food availability in C. elegans.</a> Nat Commun. 8, 14221 (2017).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NE-1000 Series of Programmable Syringe Pumps. , (2017).</li><li>Tan, M. W., Mahajan-Miklos, S., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+of+Caenorhabditis+elegans+by+Pseudomonas+aeruginosa+used+to+model+mammalian+bacterial+pathogenesis.">Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis.</a> Proc Natl Acad Sci U S A. 96 (2), 715-720 (1999).</li><li>Kim, D. 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=A+conserved+p38+MAP+kinase+pathway+in+Caenorhabditis+elegans+innate+immunity.">A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity.</a> Science. 297 (5581), 623-626 (2002).</li><li>Troemel, E. R., 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=p38+MAPK+regulates+expression+of+immune+response+genes+and+contributes+to+longevity+in+C.+elegans.">p38 MAPK regulates expression of immune response genes and contributes to longevity in C. elegans.</a> PLoS Genet. 2 (11), 183 (2006).</li><li>Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M., Troemel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bZIP+transcription+factor+zip-2+mediates+an+early+response+to+Pseudomonas+aeruginosa+infection+in+Caenorhabditis+elegans.">bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 107 (5), 2153-2158 (2010).</li><li>Byerly, L., Cassada, R. C., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+the+nematode+Caenorhabditis+elegans:+I.+Wild-type+growth+and+reproduction.">The life cycle of the nematode Caenorhabditis elegans: I. Wild-type growth and reproduction.</a> Dev Biol. 51 (1), 23-33 (1976).</li><li>Chalancon, G., 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=Interplay+between+gene+expression+noise+and+regulatory+network+architecture.">Interplay between gene expression noise and regulatory network architecture.</a> Trends Genet. 28 (5), 221-232 (2012).</li><li>Sanchez, A., Choubey, S., Kondev, J. <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+noise+in+gene+expression.">Regulation of noise in gene expression.</a> Annu Rev Biophys. 42, 469-491 (2013).</li><li>Norman, T. M., Lord, N. D., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Switching+of+Cell+Fate+in+Microbes.">Stochastic Switching of Cell Fate in Microbes.</a> Annu Rev Microbiol. 69, 381-403 (2015).</li><li>Gardner, T. S., di Bernardo, D., Lorenz, D., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+genetic+networks+and+identifying+compound+mode+of+action+via+expression+profiling.">Inferring genetic networks and identifying compound mode of action via expression profiling.</a> Science. 301 (5629), 102-105 (2003).</li><li>Samuelson, A. V., Carr, C. E., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+activities+that+mediate+increased+life+span+of+C.+elegans+insulin-like+signaling+mutants.">Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants.</a> Genes Dev. 21 (22), 2976-2994 (2007).</li><li>Edwards, C. B., Copes, N., Brito, A. G., Canfield, J., Bradshaw, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malate+and+Fumarate+Extend+Lifespan+in+Caenorhabditis+elegans.">Malate and Fumarate Extend Lifespan in Caenorhabditis elegans.</a> PLoS ONE. 8 (3), 58345 (2013).</li><li>Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R., Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans II. , (1997).</li><li>Churgin, 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=Longitudinal+imaging+of+Caenorhabditis+elegans+in+a+microfabricated+device+reveals+variation+in+behavioral+decline+during+aging.">Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging.</a> eLife. 6, 26652 (2017).</li><li>Scholz, M., Lynch, D. J., Lee, K. S., Levine, E., Biron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scalable+method+for+automatically+measuring+pharyngeal+pumping+in+C.+elegans.">A scalable method for automatically measuring pharyngeal pumping in C. elegans.</a> J Neurosci Methods. 274, 172-178 (2016).</li><li>Scholz, M., Dinner, A. R., Levine, E., Biron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+feeding+dynamics+arise+from+the+need+for+information+and+energy.">Stochastic feeding dynamics arise from the need for information and energy.</a> Proc Natl Acad Sci U S A. 114 (35), 9261-9266 (2017).</li></ol>

著者が明らかに何もありません。

マイクロ ツールは、ワームの勉強に複数の利点を提供します。PDMS のイメージング デバイスは標準 NGM 寒天プレートと比較してより高い画像品質を提供しています。複数の画像は、伝統的な方法で動物をプレートから選んだし、イメージングのための顕微鏡スライドのマウントとは異なり、1 つのワームから取得できます。さらに、定数または正確な環境の構成と動物の応答間のマッピング?...

環境条件の変化は、誘導と特定の遺伝子1,2発現の抑制を伴う遺伝的プログラムの活性化につながる可能性があります。これらの運動の変化組織同じ動物と異なる動物間の間で変数があります。このような遺伝的プログラムの研究したがって個々 の動物の縦画像および環境条件の正確な動的制御を提供するメソッドを呼び出します。
近年、マイクロ流体デバイスの応答とワーム、はえ、水クマより3,4,5,6などの小動物の行動の多くの側面を研究に使用されています。 7。アプリケーションがあります、たとえば、深い表現、光の化学刺激に対する応答における神経活動の記録と歩行などポンプ8,9,10モーター動作の追跡,11。
マイクロ ベースのアプローチは、環境手がかり、ローカルの微小のメンテナンスを可能にする柔軟な設計の正確な動的制御などへの応答の長期縦断的イメージングに恩恵を受ける可能性があります多くのプロパティを保持します。別の四分の一、およびイメージングのための有利な属性で個々 の動物。しかし、彼らの幸福に有害影響を最小限で長い時間のため流体チャンバーで動物を維持するは、実験の実行だけでなく、マイクロ流体デバイスの設計で特に注意を必要とする課題です。
ここで我々 は WormSpa、線虫の縦断的イメージングのためのマイクロ流体デバイスの使用を示します。5個々 のワームが部屋に閉じ込められました。ワームは、十分に供給し十分に健康を維持し、ストレスを軽減するために積極的、チャンバーの構造によって、ワームの卵を産むは液体や細菌懸濁液の定低流量を保証します。デザインのシンプルさと WormSpa の操作は、自分の研究計画にこのデバイスを組み込むマイクロ流路中は以前の経験を持つ研究者をようにします。

<table><tbody><tr><td>WormSpa</td><td>N/A</td><td>N/A</td><td>The CAD file for WormSpa is available from the Levine lab.</td></tr><tr><td>Compound Microscope</td><td>Zeiss</td><td>AxioObserver Z1</td><td>An inverted fluorescence microscope with a motorized stage</td></tr><tr><td>Syringe Pump</td><td>New Era Pump Systems</td><td>NE-501</td><td></td></tr><tr><td>Tubing</td><td>SCI Scientific Commodities Inc.</td><td>BB31695-PE/5</td><td>0.034&amp;rdquo; (0.86 mm) I.D. x 0.052&amp;rdquo; (1.32 mm) O.D</td></tr><tr><td>Syringe Tip</td><td>CMLsupply</td><td>901-20-050</td><td>20 Gauge x 1/2&amp;rdquo; blunt tip stainless steel canula</td></tr><tr><td>Syringe Filter</td><td>PALL</td><td>4650</td><td>Acrodisc 32 mm Syringe Filter with 5 um Supor Membrane</td></tr><tr><td>Syringe</td><td>Qosina</td><td>C3307</td><td>10 mL Male Luer Lock Syringe</td></tr><tr><td>3 Way Valve</td><td>ColeParmer</td><td>FF-30600-23</td><td>Large-bore 3-way, male-lock, stopcocks, 10/pack, Non-sterile</td></tr><tr><td>Dowel Pin</td><td>McMaster-Carr</td><td>90145A317</td><td>18-8 Stainless Steel Dowel Pins (1/32&quot; Dia. x 1/2&quot; Lg.)</td></tr><tr><td>Low Binding Microcentrifuge Tube</td><td>Corning</td><td>CL S3206</td><td>0.65 mL low binding snap cap microcentrifuge tube</td></tr></tbody></table>

a microfluidic platform for longitudinal imaging in caenorhabditis elegans

過去 10 年間でマイクロ流体技術小動物、線虫、線虫などの研究に適用されているし、便利なライブ イメージング プラットフォームの正確な制御のための機能を提供すると役に立つことがわかりましたリアルタイムでの実験条件。この記事では、WormSpa、以前発行したカスタム マイクロ流体デバイスを用いた個々 のワームのライブ イメージングを示しています。デバイスに複数のワームは個別に分離室、さまざまな生物学的プロセスの多重縦監視を許可に限定されます。機能を示すためには、病原性細菌を持つデバイスに感染していたワームと産卵および免疫応答遺伝子発現の動態は個人で継続的に監視された原理実証実験を行った動物。シンプルなデザインとこのデバイスの操作では、マイクロ流路を用いた実験で以前経験がないユーザーに適してください。このアプローチは明確に定義された条件の下で生物学的過程の縦断的観察に興味がある多くの研究者に有用であることを提案します。

プロトコルで説明されているようにデバイスに読み込まれた (46 時間ポスト L1 幼虫逮捕 25 ° C で)12歳同期の若い大人ワーム。ワームは、病原体に対する動物の反応の縦断的測定を有効にする別のチャネルで個別に位置していた。実験が成功したら、ほとんどのワームは、実験の期間の彼らのチャネルに残る。この場合、動物を積極的に検索する必要?...

Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

カエノラブディティス ・ エレガンスにおける縦断的イメージングのためのマイクロ流体プラットフォーム

この記事では、カスタム マイクロ流体デバイスを用いた個々 のワームのライブ イメージングを示しています。デバイスに複数のワームは個別に分離室、さまざまな生物学的プロセスの多重縦監視を許可に限定されます。

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

a-microfluidic-platform-for-longitudinal-imaging-caenorhabditis

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

6.4K ビュー.  Kongju National University. この記事では、カスタム マイクロ流体デバイスを用いた個々 のワームのライブ イメージングを示しています。デバイスに複数のワームは個別に分離室、さまざまな生物学的プロセスの多重縦監視を許可に限定されます。

ビデオ: A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

のためのシンプルなマイクロ流体デバイス<em生体内で&gt;</emの&gt;イメージング<em&gt; Cエレガンス</em&gt;<em&gt;ショウジョウバエ</em&gt;とゼブラフィッシュ

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

生物工学

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

のオンデマンド定量分析のためのマイクロ流体ベースの走電性<em&gt;線虫（Caenorhabditis elegans）</em&gt; &#39;ロコモーション

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

等方性光シート顕微鏡と自動細胞系譜分析による、細胞内分解能を Caenorhabditis elegans 胚発生

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

発生生物学

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

の長期カルシウムイメージングのためのアガロースマイクロチャンバー<em&gt;線虫（Caenorhabditis elegans）</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

神経科学

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

イメージング C.エレガンス</em落射蛍光顕微鏡とオープンソースソフトウェアを使用して>胚

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

生物学

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

適応マイクロ嗅覚を用いた男性c. の Elegans頭ニューロンのフェロモン応答における神経活動のイメージング

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

マイクロ流体デバイスを用いた機械的刺激と線虫の高解像度イメージング

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

縦断的蛍光モニタリングと嫌悪的介入のための固体培地上での個々の カエノラブディティスエレガンスの 長期培養

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

カエノラブディティス・エレガンスにおける縦断的イメージングのためのマイクロ流体プラットフォーム

要約

さらに動画を探す

この動画の章

のためのシンプルなマイクロ流体デバイス

のオンデマンド定量分析のためのマイクロ流体ベースの走電性

等方性光シート顕微鏡と自動細胞系譜分析による、細胞内分解能を Caenorhabditis elegans 胚発生

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

の長期カルシウムイメージングのためのアガロースマイクロチャンバー

イメージング C.エレガンス胚

適応マイクロ嗅覚を用いた男性c. の Elegans頭ニューロンのフェロモン応答における神経活動のイメージング

マイクロ流体デバイスを用いた機械的刺激と線虫の高解像度イメージング

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

縦断的蛍光モニタリングと嫌悪的介入のための固体培地上での個々のカエノラブディティスエレガンスの長期培養

カエノラブディティス ・ エレガンスにおける縦断的イメージングのためのマイクロ流体プラットフォーム

要約

さらに動画を探す

この動画の章

のためのシンプルなマイクロ流体デバイス

のオンデマンド定量分析のためのマイクロ流体ベースの走電性

等方性光シート顕微鏡と自動細胞系譜分析による、細胞内分解能を Caenorhabditis elegans 胚発生

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

の長期カルシウムイメージングのためのアガロースマイクロチャンバー

イメージング C.エレガンス胚

適応マイクロ嗅覚を用いた男性c. の Elegans頭ニューロンのフェロモン応答における神経活動のイメージング

マイクロ流体デバイスを用いた機械的刺激と線虫の高解像度イメージング

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

縦断的蛍光モニタリングと嫌悪的介入のための固体培地上での個々の カエノラブディティスエレガンスの 長期培養

カエノラブディティス・エレガンスにおける縦断的イメージングのためのマイクロ流体プラットフォーム

縦断的蛍光モニタリングと嫌悪的介入のための固体培地上での個々のカエノラブディティスエレガンスの長期培養