Ringraziamo Daniel Chen dalla Digital Video produzione Team di Buchholz High School per assistenza nell'editing del video. Questa ricerca su cellule di guardia stomatica, proteomica e metabolomica in laboratorio Chen è stata sostenuta da sovvenzioni dal US National Science Foundation (0818051, 1158000 e 1412547). Wenwen Kong è supportato dal Consiglio di borsa di studio di Cina. Dr. Qiuying Pang è supportato dalla Cina Scholarship Council e la Fondazione nazionale di scienze naturali della Cina (31570396).

1. coltivazione di piante
<ol><li>Germinare i semi in miscela di impregnazione. Crescere le piante in una camera di crescita con un'intensità luminosa di 140 µmol fotoni m− 2 s − 1 e un fotoperiodo di luce 8 h a 22 ° C e h 16 scuro a 18 ° C per 2 settimane.</li>
	<li>Trapiantare le piantine di simili dimensioni singolarmente in vasi di diametro 4" contenente il terreno e crescere per un ulteriore 3 settimane nelle stesse condizioni descritte al punto 1.1. Nota: Acqua piante con acqua del rubinetto due volte a settimana per mantenere il terreno umido.</li>
</ol>2. preparazione di stomi arricchiti
<ol><li>Rimuovere le foglie delle piante di a. thaliana 5 - settimana-vecchi individuali con un bisturi.</li>
	<li>Collegare ogni foglia a due pezzi di nastro adesivo Scotch trasparente, con un unico pezzo aderendo al lato abbagliai (inferiore) e l'altro pezzo aderendo al lato adassiale (superiore) della foglia. Lasciare il nastro sulla foglia per 5 s.</li>
	<li>Delicato peeling apart i due pezzi di nastri utilizzando il dito indice e il pollice di ogni mano per separare il lato abbagliai con pavimentazione e cellule di guardia e il lato adassiale con cellule del mesofillo.</li>
	<li>Posto le bucce dal lato abbagliai delle foglie in 60 mL di stomi apertura del buffer (50 mM KCl, 10mm MES-KOH, regolata a pH 6.2 con 1m KOH) in dimensioni 40 mm × 12 mm Petri piatti come sono stati raccolti.<ol>
<li>Ripetere i passaggi precedenti fino a quando tutte le bucce di esempio vengono raccolti.</li>
	</ol>
</li>
	<li>Posizionare le capsule di Petri contenente le bucce nella camera di crescita in condizioni di luce indicato al punto 1.1. Lasciare le bucce sotto la luce per 2 h aprire completamente gli stomi.</li>
	<li>Bucce di posto in un 150 mm x 20 mm di Petri contenente 50 mL di parete cellulare digerendo miscela di enzimi (0,7% cellulasi R-10, 0.025% macerozyme R-10, 0,1% (p/v) polivinilpirrolidone-40 e albumina di siero bovino di 0.25% (p/v) in soluzione basica di 55% (sorbitolo 0,55 M, 0.5 mM CaCl2, 0,5 mM MgCl2, acido ascorbico 0,5 mM, 10 µM KH2PO4, 5mm 4-morpholineethanesulfonic acido (MES) a pH 5.7 regolato con 1m KOH).</li>
	<li>Agitare le bucce su un agitatore reciproco in una capsula di Petri a 50 rpm per 20 min.</li>
	<li>Rimuovere bucce da enzima soluzione con una pinzetta dopo 20 min. trasferire le bucce a una dimensione 150 x 20 mm piastra Petri contenente 50 mL di acqua.</li>
	<li>Utilizzando una pipetta di trasferimento, sciacquare bucce per 15 s due volte con 10 mL di acqua per rimuovere qualsiasi soluzione enzimatica residua.</li>
	<li>Utilizzare una pinzetta per posizionare le bucce in una capsula di Petri dimensioni 150 x 20 mm con 50 mL di stomi freschi apertura del buffer come utilizzata nel passaggio 2.4. Incubare per 1 h in condizioni di luce indicato nel passaggio 1.1 per consentire gli stomi recuperare.</li>
</ol>3. l'acido abscissico (ABA) trattamento e analisi del movimento di stomi
<ol><li>In una capsula Petri dimensioni 150 x 20 mm. Incubare le bucce di stomi arricchito a 30° C per 15 min in 50 mL di tampone apertura stomatica o in 50 mL di tampone apertura stomatica con una concentrazione finale di 10 µM ABA per il tempo di trattamento appropriato.</li>
	<li>Bucce di posto su un agitatore a 50 giri/min per 5, 15 e 30 min. Dopo ogni intervallo di tempo rimuovere una buccia con le pinzette e seguire la procedura di imaging in fase 3.3.</li>
	<li>Acquisire le immagini degli stomi prima del trattamento ABA e nei vari punti di tempo dopo il trattamento ABA con un microscopio ottico.<ol>
<li>Prendete una buccia e posto su un vetrino per microscopio.</li>
		<li>Porre il vetrino sul palco di microscopio e regolare la fase, in modo che l'immagine della buccia può essere visualizzato.</li>
		<li>Regolare la multa e messa a fuoco il microscopio per ottenere un'immagine chiara delle cellule guardia del corso.</li>
		<li>Prendere diverse immagini di multipli stomi a 40 ingrandimenti.</li>
	</ol>
</li>
	<li>Misura 60 aperture stomatica usando ImageJ8.</li>
	<li>Calcolare l'errore standard e il significato a un valore di p < 0,05 di 60 misurazioni di apertura stomatica.</li>
</ol>4. proteine estrazione e separazione di SDS-Page
<ol><li>Macinare le bucce per 15 s in abbastanza azoto liquido per coprire le bucce con un refrigerate mortaio e pestello.</li>
	<li>Per ogni 50 bucce, aggiungere 3 mL di fenolo saturato di Tris (pH 8,8) e 3 mL di tampone di estrazione della proteina (0,9 M saccarosio, 0.1 M Tris-HCl, EDTA 0,01 M, 0,4% 2-mercaptoetanolo, 10 µ l di inibitore di proteasi e fosfatasi 1 mM) con una pipetta nel mortaio e poi macinare le bucce per addi 5 minuti di tradizionali in una cappa aspirante.</li>
	<li>L'estratto usando una pipetta di trasferimento e le bucce con pinzette metalliche per un Oakridge tubo centrifugo e agitano su un agitatore a 50 giri/min per 1 h a 4 ° C.</li>
	<li>Rimuovere le bucce dal tubo Oakridge centrifuga e centrifugare l'estratto a 5.000 x g per 10 min. Quindi trasferire la fase fenolica superiore ad un nuovo tubo pulito micro-centrifuga usando una pipetta.</li>
	<li>Precipitato il fenolo estratte proteine aggiungendo 5 volumi di acetato di ammonio 0.1 M ghiacciata in metanolo al 100%. Vortex brevemente e incubare per una notte a-20 ° C.</li>
	<li>Centrifugare a 20.000 x g a 4 ° C per 20 min, decantare il supernatante versando lentamente fuori dal tubo e conservare il pellet di proteina nel tubo.</li>
	<li>Lavare la pallina di proteine due volte con acetato di ammonio 0.1 M in metanolo e poi due volte con 80% di acetone e una volta con 100% di acetone freddo. Nota: Lavaggi sono fatto aggiungendo 10 mL di reagente specificato al tubo della proteina. Agitare su un agitatore a 50 rpm per 5 min, seguita da centrifugazione a 15.000 x g a 4 ° C per 10 min. Poi versare il reagente fuori dal tubo mantenendo solo il pellet di proteina.</li>
	<li>Aggiungere 1 mL di acetone freddo 100% per il pellet e risospendere pipettando lentamente su e giù.</li>
	<li>Sospensione della proteina di trasferimento tramite pipetta a una micro-centrifuga 2 mL poi Centrifugare a 20.000 x g a 4 ° C per 5 min.</li>
	<li>Rimuovere l'acetone di decantazione e dal tubo e asciugare il pellet in cappa per 10 min.</li>
	<li>Sciogliere la proteina in una provetta da micro-centrifuga con 200 µ l di tampone di dissoluzione (8m urea, 0,5% SDS, 30 mM Tris-HCl a pH 8,5) e vortex per 30 min. centrifuga a 20.000 x g a 15 ° C per 20 min e raccogliere il surnatante in un nuovo tubo.</li>
	<li>Quantificare la concentrazione nella proteina seguendo il protocollo di quantificazione della proteina9.</li>
	<li>Utilizzare campioni della proteina per la separazione delle proteine mediante elettroforesi in gel seguendo il protocollo n.10.</li>
</ol>

<ol>
	<li>Acharya, B., Assmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+interactions+in+stomatal+function.">Hormone interactions in stomatal function.</a> Plant Molecular Biology. 69 (4), 451-462 (2009).</li><li>Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., Waner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+cell+signal+transduction.">Guard cell signal transduction.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 52, 627-658 (2001).</li><li>Obulareddy, N., Panchal, S., Melotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+Cell+Purification+and+RNA+Isolation+Suitable+for+High-Throughput+Transcriptional+Analysis+of+Cell-Type+Responses+to+Biotic+Stresses.">Guard Cell Purification and RNA Isolation Suitable for High-Throughput Transcriptional Analysis of Cell-Type Responses to Biotic Stresses.</a> Molecular Plant-Microbe Interactions. 26 (8), 844-849 (2013).</li><li>Pandey, S., Wang, X. Q., Coursol, S. A., Assmann, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+applications+of+Arabidopsis+thaliana+guard+cell+protoplasts.">Preparation and applications of Arabidopsis thaliana guard cell protoplasts.</a> New Phytologist. 153 (3), 517-526 (2002).</li><li>Schroeder, J. I., Raschke, K., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+Dependence+Of+K++Channels+In+Guard-Cell+Protoplasts.">Voltage Dependence Of K+ Channels In Guard-Cell Protoplasts.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (12), 4108-4112 (1987).</li><li>Wu, F. 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=Tape-Arabidopsis+Sandwich+-+a+simpler+Arabidopsis+protoplast+isolation+method.">Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method.</a> Plant Methods. 5, 10 (2009).</li><li>Svozil, J., Gruissem, W., Baerenfaller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+targeting+of+proteins+in+Arabidopsis+leaf+mesophyll,+epidermal+and+vascular+tissues.">Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.</a> Frontiers in Plant Science. 6, (2015).</li><li>Schneider, C., Rasband, W., Eliceiri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Agnew, B., Murray, D., Patton, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+solid-phase+fluorescence-based+protein+assay+for+quantitation+of+protein+electrophoresis+samples+containing+detergents,+chaotropes,+dyes,+and+reducing+agents.">A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents.</a> Electrophoresis. 25 (15), 2478-2485 (2004).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laemmli-SDS-PAGE.">Laemmli-SDS-PAGE.</a> Bio-protocol. Bio101 (80), (2011).</li><li>Thompson, 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=Tandem+mass+tags:+A+novel+quantification+strategy+for+comparative+analysis+of+complex+protein+mixtures+by+MS/MS+(vol+75,+pg+1895).">Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS (vol 75, pg 1895).</a> Analytical Chemistry. 78 (12), 4235-4235 (2006).</li><li>Wiese, S., Reidegeld, K. A., Meyer, H. E., Warscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+labeling+by+iTRAQ:+A+new+tool+for+quantitative+mass+spectrometry+in+proteome+research.">Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research.</a> Proteomics. 7 (3), 340-350 (2007).</li></ol>

Conflitti di interesse non dichiarati.

Cellule di guardia sono un sistema modello per studiare i meccanismi di trasduzione del segnale nelle piante ed è importante garantire che la preparazione dei campioni utilizzati nello studio è il più appropriato per rispondere alle domande biologiche. Nonostante il crescente interesse in cellule di guardia all'interno della comunità di ricerca di pianta, non esiste un metodo universale su come preparare stomatica cellule di guardia che permettesse sia il movimento stomatica e fisiologia così come i cambiamenti mole...

Gli stomi giocano un ruolo importante nella fisiologia complessiva e fitness delle piante. Queste strutture specifiche di pianta molto piccola sono formate da due cellule epidermiche specializzate, conosciute come cellule di guardia e si trovano più abbondantemente sulla superficie abbagliai delle foglie. Gli stomi sono necessari per lo scambio di gas tra la pianta e l'atmosfera, nonché il controllo della perdita di acqua attraverso la traspirazione. Cellule di guardia regolare apertura stomi attraverso l'integrazione di diversi esterni (ad es., luce, umidità, contatto agente patogeno, livelli di anidride carbonica) e interni (ad es., ormoni endogeni) stimoli1,2. Negli ultimi 20 anni, questo tipo di cella è diventato un sistema modello per studiare i processi negli impianti di segnalazione delle cellule. Queste cellule costituiscono una piccola frazione delle cellule totali in una foglia; così, per studiare il loro cellulare unico, proprietà biochimiche e molecolari in maniera singola cellula, è necessario isolarli dagli altri tipi di cella foglia. In passato, gli studi delle cellule di guardia hanno coinvolto spesso la preparazione di cellule di guardia protoplasti (GCP)3,4,5. Questo in genere richiede l'utilizzo di grandi quantità di enzimi degradanti della parete cellulare e/o rottura meccanica tramite fusione e ha dimostrato di essere costoso e richiede tempo, come in genere occorrono molte ore per preparare un campione GCP, spesso volte con poca resa. Ancora più importante, poiché le cellule di guardia fungono da coppie complete di regolare apertura stomi, l'uso di GCP può essere visto come un sistema artificiale per studiare segnalazione delle cellule di guardia.
Qui, abbiamo sviluppato un metodo per preparare gli stomi in cui cellule intatte di guardia sono arricchite e mantenere risposte fisiologiche agli stimoli. Questo metodo è stato ispirato da un metodo che è stato usato per isolare mesofillo cellula protoplasti6,7. Nel nostro metodo, gli stomi sono preparati utilizzando chiaro nastro Scotch per separare prima la maggioranza delle cellule del mesofillo attaccato allo strato adassiale della foglia dallo strato abbagliai contenente la pavimentazione e le cellule di guardia. Questo viene fatto da un pezzo di nastro per ogni lato della foglia di apposizione e sbucciarle apart. Le cellule dallo strato abbagliai sono ammessi per recuperare sotto la luce in un buffer di stomi-apertura. Successivamente le cellule restanti di pavimentazione vengono rimossi utilizzando una piccola quantità di parete cellulare, enzimi, lasciando dietro di sé delle cellule di guardia che si arricchiscono sul nastro degradativi.
In questo semplice metodo, stomatica cellule di guardia sia valide e reattivo può rapidamente e in modo efficiente preparatevi, prendere meno di un'ora per ottenere cellule di guardia stomatica arricchite da 50 bucce con un costo minimo. Il metodo qui impone meno danni per le cellule della pianta rispetto ai metodi precedenti dove sono preparati i protoplasti. Inoltre, materiali raccolti da questa quantità di proteina auspicabile resa metodo. La cosa più importante, perché la foglia non è sottoposto a fusione o tempi di digestione lunga e cellule di guardia rimangono intatte, i risultati degli studi sarà più strettamente pertinenti a quella di biologia naturale di una cella di guardia. Con questo metodo, stomatici movimenti possono essere correlati in tempo reale con i cambiamenti a livello molecolare. Così, le conoscenze acquisite dagli studi utilizzando questo metodo di preparazione sarebbe importante per una comprensione più completa di segnalazione delle cellule di guardia.
Abbiamo usato la pianta Arabidopsis thaliana come modello; Tuttavia questo protocollo può essere applicato all'arricchimento delle cellule di guardia stomatica in altre specie di piante quali Brassica napus con piccole modifiche nel tempo di digestione. Come un proof of concept, abbiamo dimostrato che cellule di guardia stomatica arricchite tramite questo metodo sono vitali e sensible a reagire agli stimoli come mostrato dalla fluoresceina diacetato (FDA) analisi e studi che utilizzano l'ormone vegetale l'acido abscissico (ABA), rispettivamente. Inoltre, abbiamo dimostrato che le proteine di alta qualità possono essere isolate da queste cellule di guardia. Qui, descriviamo un protocollo dettagliato di questo processo.

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			<td height="21" style="height:21px;width:307px;">Stainless Steel Surgical Scalpel</td>
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			<td style="width:119px;">&#160;NC9999403</td>
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			<td height="21" style="height:21px;width:307px;">Scotch Tape (3M)</td>
			<td style="width:297px;">ULINE</td>
			<td style="width:119px;">S-9781</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">Petri Dish</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td>BR455701</td>
			<td></td>
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			<td height="21" style="height:21px;">Cellulase (Onozuka R-10)&#160;</td>
			<td>Yakult Pharmaceutical Industry Co., Ltd.</td>
			<td>21560003-3</td>
			<td></td>
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			<td height="21" style="height:21px;">Macerozyme R-10</td>
			<td>Yakult Pharmaceutical Industry Co., Ltd.</td>
			<td>21560003-4</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">DM6000B Microscope&#160;</td>
			<td>Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Protease and Phosphatase Inhibitor Cocktails</td>
			<td style="width:297px;">Thermo Fisher Scientific&#160;</td>
			<td>&#160;PI78443</td>
			<td></td>
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			<td height="22" style="height:22px;">Oak Ridge High-Speed PPCO Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>3119-0030</td>
			<td></td>
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			<td height="21" style="height:21px;">EZQ Protein Quantitation Kit</td>
			<td style="width:297px;">Thermo Fisher Scientific</td>
			<td>R33200</td>
			<td></td>
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			<td height="21" style="height:21px;">Fluorescein Diacetate (FDA)</td>
			<td style="width:297px;">Thermo Fisher Scientific</td>
			<td>F1303</td>
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			<td height="21" style="height:21px;">Abscisic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4906</td>
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			<td height="21" style="height:21px;width:307px;">Metro Mix 500</td>
			<td style="width:297px;">BWI Companies&#160;</td>
			<td style="width:119px;">TX-500</td>
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			<td height="21" style="height:21px;width:307px;">Laemmli Sample Buffer</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">161-0737</td>
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			<td height="21" style="height:21px;width:307px;">Bio-Safe Comassie (G-250)</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">161-0786</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">Microcentrifuge Tube (2mL)</td>
			<td style="width:297px;">USA Scientific, Inc.</td>
			<td style="width:119px;">1620-2700</td>
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			<td height="21" style="height:21px;width:307px;">ImageJ software</td>
			<td style="width:297px;">National Institute of Health</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">Tweezers&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4017-1EA</td>
			<td></td>
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			<td height="21" style="height:21px;width:307px;">12% Mini-PROTEAN TGX Precast Gel</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">456-1043</td>
			<td></td>
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			<td height="21" style="height:21px;">Microscope slides</td>
			<td style="width:297px;">Fisherbrand</td>
			<td>12-550-A3</td>
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stomata tape-peel: an improved method for guard cell sample preparation

Lo studio delle cellule di guardia è essenziale per la conoscenza dei contributi specifici di questo tipo di cella alla funzione generale delle piante. Tuttavia, è spesso difficile isolare e così loro studio spesso rappresenta una sfida.
Questo studio stabilisce un metodo di arricchimento stomatica delle cellule di guardia. Il protocollo utilizza nastro Scotch per isolare cellule di guardia ed estrarre le proteine dalle cellule. Questo metodo migliora l'integrità e il rendimento della cella di guardia durante l'isolamento e fornisce un campione affidabile per lo studio dei processi di segnalazione delle cellule e come essi correlare ai fenotipi movimento stomatica. In questo metodo, le foglie delle piante erano divise tra i due porzioni di nastro e pelate a pezzi per rimuovere il lato abbagliai. Questo protocollo è stato applicato al tessuto di foglia di Arabidopsis per isolare cellule di guardia. Le cellule sono state trattate con diacetato di fluoresceina (FDA) per determinare la vitalità e l'acido abscissico (ABA) per valutare il movimento di stomi in risposta all'ABA. In conclusione, il presente protocollo si rivela utile per la preparazione di cellule di guardia stomatica arricchite e isolare proteine. La qualità delle cellule e proteine ottenute qui enable fisiologico e altri studi biologici.

Un'immagine rappresentativa di Arabidopsis cellule di guardia prima e dopo la digestione del mesofillo e cellule epidermiche è illustrata nella Figura 1. Attuabilità delle cellule di guardia prima e dopo la rimozione del mesofillo e cellule epidermiche possono essere osservate utilizzando FDA per misurare l'attività enzimatica e l'integrità della membrana delle cellule (Figura 1B e 1D). L...

Watch this Scientific Journal Video about Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation at JoVE.com

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

Questo protocollo descrive un metodo di preparazione arricchite stomatica cellule di guardia che è utile per gli studi biologici fisiologici e altri.

Gli stomi nastro-Peel: Un metodo migliore per la preparazione del campione di cellule di guardia

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

stomata-tape-peel-an-improved-method-for-guard-cell-sample-preparation

Research

JoVE Journal

Biology

14.9K Views.  University of Florida. Questo protocollo descrive un metodo di preparazione arricchite stomatica cellule di guardia che è utile per gli studi biologici fisiologici e altri.

Video: Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Preparazione del campione: il metodo ultra sottile strato

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Ricerca

Biologia

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Assemblaggio, carico, allineamento e di una cella analitica del campione Ultracentrifuga

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

Un metodo migliorato di isolamento dell&#39;RNA da Pino Loblolly ( P. taeda L.) e altre specie di conifere

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Un metodo migliore per precisa e rapida misurazione delle performance in volo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Preparazione del campione per microscopia a forza atomica Virion singola e imaging a fluorescenza a super risoluzione

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Miglioramento Tecnica Swiss-laminazione per intestinale Preparazione del tessuto per immunoistochimica e analisi immunofluorescenza

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Preparazione del campione per l&#39;analisi di massa Citometria

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Un approccio alternativo per la preparazione del campione con un basso numero di cellule per l&#39;analisi TEM

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Un metodo sezionamento migliorato per Recalcitrante tessuti vegetali Campioni

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

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.