Questo lavoro è stato sostenuto dal National Science Foundation (Grant no. DBI 0.519.984) e il National Institutes of Health (Grant no. 5R01GM078279) ..

1) Preparazione del materiale vegetale <ol><li> Protoplasti può essere derivato da molte specie vegetali e tessuti diversi, a condizione che il giusto mix di parete cellulare enzimi è utilizzato 1. Prima di un vero e proprio esperimento è intrapreso, una piccola digestione del materiale è consigliabile al fine di valutare l&#39;efficienza protoplasting del tessuto, enzimi, ecc e per stimare la percentuale delle cellule positive per l&#39;ordinamento delle cellule. Qui, protoplasti derivati ​​dalle radici di piantine di Arabidopsis thaliana, che cellule di tipo specificamente esprimere la proteina verde fluorescente (GFP) sono utilizzati. L&#39;endoderma e il centro di quiescenza sono contrassegnati da P SCR:: GFP e il centro quiescente da P WOX5:: GFP 2,3 (Figura 1). </li><li> Piantine sono cresciute in idroponica phytatrays (Sigma; Figura 2a) su un filtro di nylon (250 mesh micron; NITEX) che permette di crescere attraverso le radici nel terreno di crescita (0,22% w / v Murashige e medie Skoog Basal [sigma], 1% w / v di saccarosio, 0,05% w / v MES [2 - (N-morfolino) Acido etano], pH 5,7 con KOH). In alternativa, le piante possono essere coltivate in cima ad un filtro di nylon (100 mesh micron) in posizione verticale piastre di agar 1% (Figura 2b). </li><li> L&#39;uso dei filtri di cui sopra non solo aiuta nella raccolta delle radici, facilita anche facile trattamento aggiuntivo delle piantine, se lo si desidera. I filtri permettono le piantine da trasferire in massa per phytatrays nuovi o piastre di agar integrato con un catalizzatore di interesse. Per esempio, il set up phytatray è stato usato per cellulare di tipo specifico per l&#39;analisi della risposta trascrizionale al trattamento azoto in piantine di Arabidopsis 4. </li><li> Assicurarsi che il vostro microscopio marcatore fluorescente è correttamente espresso (soprattutto quando si utilizza l&#39;opzione di trattamento, come la cellula tipo-specifici marcatori stessi potrebbe essere influenzata dal trattamento). In questo caso, piantine sono controllati al microscopio a fluorescenza (Nikon; Figura 1). Da notare che delle cellule tipo-specifici linee marcatore fluorescente dovrebbe essere caratterizzata inizialmente sotto un microscopio confocale a determinare esattamente quali tipi di cellule sono marcati e determinano variabilità nell&#39;espressione. </li></ol> 2) Preparazione della soluzione protoplasting <ol><li> Sciogliere 1,25% w / v Cellulase (Yakult), 0,3% w / v Macerozyme (Yakult), 0,4 M D-mannitolo, 20 mM MES e 20 mM KCl (da uno stock 1 M) in acqua demineralizzata e regolare il pH a 5,7 con 1 M Tris / HCl pH 7.5. Questa soluzione sarà leggermente torbido. </li><li> Riscaldare la soluzione a 55 ° C per 10 minuti (la soluzione diventa chiara) e lasciarlo raffreddare a temperatura ambiente prima di aggiungere 0,1% w / v BSA (albumina di siero bovino), 10 mM CaCl 2, e 5 mM β-mercaptoetanolo . </li></ol> 3) La raccolta e protoplasting del materiale vegetale <ol><li> Le radici vengono raccolte a loro raschiando via la rete di nylon con un bisturi e depositati in un pallone contenente la soluzione protoplasting. In generale, 10 ml di soluzione protoplasting è utilizzato per 1.500 radici piantina. </li><li> Agitare delicatamente i palloni (75 giri) a temperatura ambiente per un&#39;ora. Un tempo di incubazione più lungo può aumentare la resa protoplasti, ma anche aggiungere l&#39;effetto di protoplasting stesso sull&#39;espressione genica. </li><li> Filtrare la soluzione protoplasti con un colino cellula 40 micron (BD Falcon) e dividere la soluzione più conica provette da 15 ml (BD Falcon). </li><li> Gira i tubi in un basculanti centrifuga per 10 minuti a 500 G. noti che questa velocità di centrifugazione dipenderà dal tipo di protoplasti utilizzati, la loro fragilità e la quantità di detriti cellulari prodotte durante il trattamento enzimatico. </li><li> Eliminare la maggior parte surnatante, risospendere il protoplasti nella soluzione rimanente e verifica di microscopio (Figura 3). </li><li> Fare uso di un emocitometro di stimare il numero e la densità di protoplasti. La densità delle cellule determina la velocità di iniezione del campione, il tasso di eventi al secondo e quindi il tempo totale necessario per ordinare le cellule al FACS (vedere anche paragrafo 4.3). </li><li> 3.7) In entrambi i casi procedere direttamente al FACS o lavare e risospendere il protoplasti in una soluzione di incubazione, come W5 (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM MES, aggiustare il pH a 5,7 con KOH) o la soluzione protoplasting senza l&#39; aggiunto enzimi. Specialmente quando esaminano i cambiamenti trascrizionali, è di fondamentale importanza per ridurre al minimo l&#39;esposizione dei campioni a condizioni che potrebbero influenzare l&#39;espressione genica, come la modifica del buffer sono conservati i protoplasti a. Si raccomanda pertanto di mantenere il protoplasti in soluzione protoplasting e procedere FACS per il più presto possibile. </li></ol> 4) Ordinamento fluorescenza cellulare attivo di protoplasti <ol><li> Accendere e preparare il cell sorter. Qui, un FACSAria (BD) è usato. </li><li> Impostazione di un flusso di flusso con un 100 &amp; mu; ugello m ed una pressione di 20 psi guaina. </li><li> La densità delle cellule e la velocità di iniezione del campione può essere regolata per il particolare esperimento basato sul fatto che una resa migliore o più veloce di velocità raggiungibile è desiderata. Abbiamo ordinati con successo con densità fino a 10.000.000 cellule / ml. </li><li> Utilizzare l&#39;opzione agitazione campione sul FACS per evitare la sedimentazione dei protoplasti. Se intasamento del FACS è un problema, ci sono tre possibili passi risoluzione dei problemi: 1. Eseguire un campione-line backflush. 2. Diluire la sospensione di protoplasti per ridurre la densità. 3. Pulire la soluzione protoplasti ripetendo la fase di filtrazione (3.3) dopo la centrifugazione e la risospensione. </li><li> Preparare l&#39;apparato per misurare la dispersione in avanti (FSC), side scatter (SSC) ed emissione a 530/30 nm per GFP e nm 610/20 per lo spettro autofluorescenza rosso (RSA), dopo l&#39;eccitazione da un laser a 488 nm. Questi sono in sostanza gli unici parametri utilizzati per isolare GFP-positive protoplasti. Qui, le regolazioni di tensione sono state le seguenti: FSC - 60V, SSC 250V, 350V GFP e RSA 335V. Notare che le impostazioni ottimali di tensione sarà diverso per ogni FACS e sarà nemmeno bisogno di essere regolata per tutta la durata del cell sorter. </li><li> Inizia la creazione di un dotplot per scatter scatter in avanti rispetto a lato. Applicare le impostazioni di tensione in modo che gli eventi misurati sono al centro del complotto. </li><li> Successivamente, creare un dot plot di segnali di fluorescenza verde contro rosso. Applicare le impostazioni di tensione in modo che gli eventi misurati resa una popolazione centrato diagonale al complotto quando si guarda una wild-type (non GFP) sospensione di protoplasti. Una sospensione di protoplasti derivata da una linea di marcatore GFP produce una popolazione chiara di verde fluorescente eventi mai visti in campioni di wild-type. </li><li> Impostare vincoli di compensazione per registrare per sovrapposizione spettrale tra GFP e RSA. Corretto le impostazioni vincolo compensazione permettere una migliore separazione della GFP-positive protoplasti dal non-GFP protoplasti e detriti. I vincoli usato qui sono le seguenti: RSA, meno 17,91% GFP. </li><li> Impostare un cancello per identificare GFP-positive eventi, un controllo negativo di non-GFP protoplasti dovrebbe essere usata per aiutare a definire i confini cancello (Figura 4). </li><li> Implementare un cutoff dispersione in avanti in modo da lasciare piccoli detriti fuori dell&#39;analisi. Visualizza la GFP-positive eventi nel complotto contro FSC SSC per aiutare a determinare il posizionamento del taglio. Qui, il cut-off è stato fissato a 5.000. Si noti che il FACS conterà detriti come ordinare gli eventi e un campione con alti livelli di detriti potrebbero avere un diverso eventi GFP positive per cento del previsto. Questo non è necessariamente un problema. Tuttavia, i detriti più nel campione, il più a lungo l&#39;ordinamento prendere. </li><li> A seconda della sperimentazione e l&#39;abbondanza del tipo di cellula da analizzare, impostare la modalità di precisione FACS sia per la resa ottimale o purezza ottimale delle cellule ordinati. </li><li> Per l&#39;estrazione di RNA, preparare tubi di raccolta (1,5 ml tubi microcentrifuga) con la giusta quantità di tampone di estrazione dell&#39;RNA. Con questa impostazione, 20.000 eventi ordinamento produrrà un volume totale di circa 100 l che sono stati ordinati in a 350 l di tampone di estrazione (RNeasy micro kit, QIAGEN). Mescolare i campioni dopo il completamento del tipo di sospensione cellulare possono mettere in cima. </li><li> Conservare i campioni o procedere direttamente alla RNA. Analisi di microarray di successo sono stati preformati con l&#39;RNA estratto da un minimo di 500 eventi ordinati. Qui, abbiamo usato un kit RNeasy estrazione micro (QIAGEN), il WT-Ovation Pico sistema di amplificazione di RNA e FL-cDNA Ovation Biotina Modulo V2 (NuGen). </li></ol> Rappresentante Risultati Un phytatray di circa 1.500 di una settimana-vecchio P SCR:: piantine GFP prodotto circa 60.000 protoplasti (misurata in emocitometro). 2,6% di 65.000 eventi FACS-trattati sono stati definiti come GFP-positive e sono stati ordinati (Fig. 4b). Otto lastre di circa 1.500 di quattro giorni di età P WOX5:: piantine GFP ogni (12.000 in totale) fruttò circa 30.000.000 protoplasti (misurata in emocitometro). 0,063% di 16.000.000 eventi FACS-trattati sono stati definiti come GFP-positive e sono state ordinate (Figura 4c). 10.000 eventi ordinati sono tipicamente utilizzati per l&#39;estrazione di RNA e può produrre 20-140 ng di RNA totale (Figura 5). <img src="/files/ftp_upload/1673/1673fig1.jpg" alt="Figura 1" /> Figura 1. Cellula tipo-specifici linee GFP marcatore nella radice di Arabidopsis. Immagini di microscopia a fluorescenza sono state prese con un contrasto di interferenza differenziale (DIC) e un GFP filtro su un microscopio Eclipse 90i (Nikon) in esecuzione su software Metamorph (Molecular Devices). Le immagini DIC e GFP sono stati sovrapposti per fini di visualizzazione. Le due linee marker usato in questo esperimento visivo vengono mostrati;a) P SCR:: GFP e b) P WOX5:: GFP. <img src="/files/ftp_upload/1673/1673fig2.jpg" alt="Figura 2" /> Figura 2. Semenzali impianto condizioni di crescita. Sono state coltivate in un controller ambientale idroponica in phytatrays (a) o su piastre di agar in posizione verticale (b). <img src="/files/ftp_upload/1673/1673fig3.jpg" alt="Figura 3" /> Figura 3. Protoplasti vegetali che esprimono GFP. Immagini di microscopia a fluorescenza sono state prese con un contrasto di interferenza differenziale (DIC) e un filtro GFP un microscopio Eclipse 90i (Nikon) in esecuzione su software Metamorph (Molecular Devices). Le immagini DIC e GFP sono stati sovrapposti per fini di visualizzazione. Le frecce indicano una cella scoppio, detriti cellulari e un GFP-positive protoplasti. La distanza tra due linee bianche è di 50 micron. <img src="/files/ftp_upload/1673/1673fig4.jpg" alt="Figura 4" /> Figura 4. Fluorescenza separazione delle cellule attivate del GFP-positive protoplasti protoplasti derivato da wild-type (a) P SCR:.: GFP (b) o P WOX5:: GFP (c) le linee marcatori sono stati analizzati e ordinati con un FACSAria (BD) utilizzando porte definito su un dotplot di verde (530/30 nm; asse x) rispetto rossa (610 / 20 nm; asse y) fluorescenza. 100.000 eventi sono presentati in ogni trama. Gli eventi che rientrano nel cancello ordinamento GFP sono evidenziati in verde. <img src="/files/ftp_upload/1673/1673fig5.jpg" alt="Figura 5" /> Figura 5. Rappresentante estrazioni di RNA da 10.000 cellule ordinati. Cellule sono state ordinate direttamente in RNA tampone di estrazione (QIAGEN), l&#39;RNA è stato purificato e controllato per la concentrazione, la purezza e l&#39;integrità su un 2100 Bioanalyzer (Agilent). Tre repliche per entrambe le linee marker sono mostrati.

<ol>
	<li>Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+maize+and+Arabidopsis+mesophyll+protoplasts.">Signal transduction in maize and Arabidopsis mesophyll protoplasts.</a> Plant Physiol. 127, 1466-1475 (2001).</li><li>Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+SCARECROW+function+reveals+a+radial+patterning+mechanism+common+to+root+and+shoot.">Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.</a> Development. 127, 595-603 (2000).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Gifford, M. L., Dean, A., Gutierrez, R. A., Coruzzi, G. M., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+nitrogen+responses+mediate+developmental+plasticity.">Cell-specific nitrogen responses mediate developmental plasticity.</a> Proc Natl Acad Sci U S A. 105, 803-808 (2008).</li><li>Bargmann, B. O. R., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+fluorescent+selection+permits+precise,+rapid,+and+in-depth+overexpression+analysis+in+plant+protoplasts.">Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.</a> Plant Physiol. 149, 1231-1239 (2009).</li><li>Petersson, S. V., Johansson, A. I., Kowalczyk, M., Makoveychuk, A., Wang, J. Y., Moritz, T., Grebe, M., Benfey, P. N., Sandberg, G., Ljung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Auxin+Gradient+and+Maximum+in+the+Arabidopsis+Root+Apex+Shown+by+High-Resolution+Cell-Specific+Analysis+of+IAA+Distribution+and+Synthesis.">An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis.</a> Plant Cell. 21, 1659-1668 (2009).</li></ol>

Protoplasti può, in linea di principio, derivato da una varietà di tessuti vegetali, ottimizzando le condizioni favorevoli permetterà di migliorare notevolmente la qualità e la quantità di RNA. Sia la soluzione protoplasting e il buffer di incubazione elettiva utilizzati influenzano questo aspetto. Molte diverse proteine ​​fluorescenti possono essere utilizzati, a seconda delle capacità del FACS utilizzato, ad esempio, GFP, RFP, YFP, CFP o le loro varianti e derivati. L&#3...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>250 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-250/50</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-100/47</td>
<td>&nbsp;</td>
<tr>
<td>Square petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-10k</td>
<td>&nbsp;</td>
<tr>
<td>Phytatrays</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1552</td>
<td>&nbsp;</td>
<tr>
<td>Murashige and Skoog Basal Medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S5-3</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2933</td>
<td>&nbsp;</td>
<tr>
<td>KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1767</td>
<td>10 M stock</td>
</tr>
<tr>
<td>Eclipse 90i microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9546</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8041</td>
<td>1 M stock</td>
</tr>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3912</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>CALBIOCHEM</td>
<td>444203</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2536</td>
<td> 1 M stock</td>
</tr>
<tr>
<td>orbital shaker</td>
<td>&nbsp;</td>
<td>LAB-LINE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>conical 15 ml tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352196</td>
<td>&nbsp;</td>
<tr>
<td>table centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>Legend RT</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>FACSAria</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20170-38</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy micro kit</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>74004</td>
<td>&nbsp;</td>
<tr>
<td>WT-Ovation Pico RNA Amplification System</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>3300_12</td>
<td>&nbsp;</td>
<tr>
<td>FL-Ovation cDNA Biotin Module V2</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>4200_12</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence activated cell sorting of plant protoplasts

Ad alta risoluzione, il tipo specifico di cellule analisi dell&#39;espressione genica migliora notevolmente la comprensione della regolazione dello sviluppo e delle risposte agli stimoli ambientali in ogni organismo multicellulare. Ibridazione in situ e la visualizzazione gene reporter può entro certi limiti essere utilizzate a tal fine, ma per alta risoluzione quantitativa RT-PCR o high-throughput a livello di analisi del trascrittoma l&#39;isolamento di RNA da particolari tipi di cellule è necessaria. Dissociazione del tessuto cellulare che esprime un marcatore fluorescente proteina in un tipo cellulare specifico e successivo ordinamento fluorescenza cellulare attivato (FACS) permette di raccogliere quantità sufficienti di materiale per l&#39;estrazione di RNA, cDNA analisi sintesi / amplificazione e microarray. Un ampio insieme di cellule tipo-specifici linee giornalista fluorescente è disponibile alla comunità di ricerca delle piante. In questo caso, due linee marcatore della radice Arabidopsis thaliana sono utilizzati: P SCR:: GFP (endoderma e centro quiescente) e P WOX5:: GFP (centro di riposo). Un gran numero (migliaia) di piantine sono cresciute idroponica o su piastre di agar e raccolte di ottenere abbastanza materiale radice per ulteriori analisi. Cellulari dissociazione di materiale vegetale è ottenuta per digestione enzimatica della parete cellulare. Questa procedura si avvale di alta osmolarità indotta plasmolysis e cellulasi disponibili in commercio, pectinasi e emicellulasi di rilasciare protoplasti in soluzione. FACS di cellule GFP-positive si avvale della visualizzazione del verde rispetto al rosso di spettri di emissione protoplasti eccitati da un laser a 488 nm. GFP-positive protoplasti si distinguono per il loro rapporto è aumentato di verde al rosso di emissioni. Protoplasti sono generalmente ordinati direttamente nel tampone di estrazione di RNA e conservati per l&#39;ulteriore elaborazione in un secondo momento. Questa tecnica si rivela essere semplice e praticabile. Inoltre, è dimostrato che può essere usata senza difficoltà per isolare un numero sufficiente di cellule per l&#39;analisi del trascrittoma, anche per tipi di cellule molto scarse (ad esempio le cellule quiescenti centro). Infine, una messa a punto di crescita per piantine di Arabidopsis è dimostrato che permette di trattare semplice delle piante prima della separazione delle cellule (ad esempio per il tipo specifico di cellule analisi delle risposte di stress biotici e abiotici). Supplementari potenziali usi per FACS di protoplasti vegetali sono discussi.

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting of Plant Protoplasts at JoVE.com

Fluorescence Activated Cell Sorting of Plant Protoplasts

Un metodo per isolare specifici tipi cellulari da materiale vegetale è dimostrata. Questa tecnica utilizza le linee marcatore transgenici che esprimono proteine ​​fluorescenti in particolari tipi di cellule, cellulari dissociazione e fluorescenza separazione delle cellule attivate. Inoltre, una configurazione di crescita è stabilito qui che facilita il trattamento delle<em&gt; Arabidopsis thaliana</em&gt; Piantine prima della separazione delle cellule.

Fluorescenza separazione delle cellule attivate delle protoplasti impianto

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

fluorescence-activated-cell-sorting-of-plant-protoplasts

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24.6K Views.  New York University. Un metodo per isolare specifici tipi cellulari da materiale vegetale è dimostrata. Questa tecnica utilizza le linee marcatore transgenici che esprimono proteine ​​fluorescenti in particolari tipi di cellule, cellulari dissociazione e fluorescenza separazione delle cellule attivate. Inoltre, una configurazione di crescita è stabilito qui che facilita il trattamento delle Arabidopsis thaliana Piantine prima della separazione delle cellule.

Video: Fluorescence Activated Cell Sorting of Plant Protoplasts

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Vescicole lipidiche-mediata Cromatografia di affinità mediante Magnetic separazione delle cellule attivate (LIMACS): un nuovo metodo per l&#39;analisi delle interazioni proteina-lipide

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Ricerca

Biologia

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeossiuridina (BrdU) Etichettatura e successive ordinamento fluorescenza cellulare attivo per la cultura indipendente Identificazione di carbonio organico disciolto-degradanti batterioplancton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Cella di analisi specifica di<em&gt; Arabidopsis</em&gt; Foglie usando fluorescenza activated cell sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Isolamento di fibroblasti normali e tumorali associata da tessuti freschi di fluorescenza activated cell sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interazioni proteina-proteina visualizzati tramite bimolecular complementazione fluorescenza in protoplasti di tabacco e foglie

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Misurare il osmotica dell&#39;acqua Permeabilità Coefficiente (P<sub&gt; F</sub&gt;) Di celle sferiche: protoplasti impianto isolato come un esempio

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Analisi Rapida di fenotipi circadiani in Arabidopsis protoplasti transfettate con un luminescenti Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizzazione Frequenza Stromule con microscopia a fluorescenza

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Luce Foglio microscopia a fluorescenza delle radici delle piante che crescono sulle superficie di un gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Magnetico-Activated Cell Sorting strategie per isolare e purificare Synovial cellule staminali mesenchimali liquido derivato da un modello di coniglio

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...