Nicotiana benthamiana piante sono coltivate in una temperatura controllata camera di crescita a 25 ° C. Completamente ampliato foglie sane di 3-6 piante settimana di vita sono utilizzati. Suggerimento: I risultati migliori si ottengono utilizzando le foglie emergenti 1. Agrobacterium protocollo di infiltrazione transitoria: Giorno 1 <ol><li> LB Streak / rifampicina (25 mg / ml) / kanamicina (100 mg / ml) piastre di agar con gli stock di glicerolo di Agrobacterium tumefaciens (ceppo LBA4404) contenente i vettori appropriato con il gene (s) per essere analizzati per la morte cellulare e il vettore contenente la cassetta GUS sotto un promotore costitutivo. Sempre includere un controllo vettoriale vuoto come controllo negativo. </li><li> Incubare a 28 ° C per 2 giorni. </li></ol> 3 ° giorno <ol><li> Inoculare 2 ml di LB contenente rifampicina (25 mg / ml) e kanamicina (100 mg / ml) con una singola colonia di ogni LB / Rifampicina / kanamicina piastra precedentemente striato. </li><li> Incubare ogni cultura agitando a 28 ° C per 24 ore a 200 rpm fino a quando la densità massima crescita è stata raggiunta. </li></ol> Suggerimento: Agglomerante a volte osservato, in cui le culture caso devono essere accuratamente risospesa (cioè pipettando su e giù) prima di procedere. 4 ° giorno <ol><li> Dopo l&#39;incubazione, aumentare il volume totale di 10 ml di LB fresca (contenente gli antibiotici del caso) e acetosyringone (concentrazione finale 25 mM). </li><li> Incubare ogni cultura agitando a 28 ° C per altre 16 ore. </li></ol> 5 ° giorno <ol><li> Il giorno seguente lavare ogni cultura due volte con l&#39;aggiunta di 10 ml (non aggiungere acetosyringone) medio infiltrazione (10 mM MgSO 4 0,7 H 2 O, 9 mM MES, pH 5.6) e centrifugare a 4000 xg per 10 minuti a temperatura ambiente. </li><li> Dopo la fase di centrifugazione finale, risospendere ogni cultura in 5 ml di mezzo di infiltrazione contenente acetosyringone (100 mM). </li><li> Misurare il diametro esterno 600 nm, misura tra 0,1 e 0,9 sono accettabili. Suggerimento: OD 600nm di fino a 0,1 sono stati utilizzati senza un problema, tuttavia, più elevati OD può produrre risultati più consistenti. </li><li> Incubare le culture per ulteriori 3 ore per preparare le culture Agrobacterium di infezione. </li><li> Culture miscela contenente il gene (s) da analizzare e il controllo negativo (Agrobacterium contenente il vettore vuoto) in un rapporto 1:1 con la cultura che contiene la cassetta GUS. </li><li> Infiltrarsi nel abaxial (sotto) lato di nuova lascia emergere con un 1 ml ago-meno siringa (Fig. 1). Suggerimento: Infiltrati sia la miscela di controllo negativo (vuoto vettore + cassetta GUS) e la miscela contenente il gene da testare (gene x + cassetta GUS) su foglie opposte della stessa pianta. Utilizzare impianti triplice copia per ogni trattamento. </li><li> 3 giorni dopo l&#39;infiltrazione delle accise infiltrato foglie e analisi per l&#39;espressione delle proteine ​​GUS. </li></ol> 2. Istochimiche GUS test 8 ° giorno <ol><li> Vuoto infiltrarsi nella X-Gluc medio substrato nelle foglie escisse. </li><li> Incubare al buio a temperatura ambiente per una notte o fino distinte macchie blu. </li></ol> Giorno 9 <ol><li> Risciacquare con acqua distillata. </li><li> Incubare in etanolo al 70% fino a clorofilla viene rimosso, poi il trasferimento di acqua distillata di nuovo. Visivamente valutare i livelli di espressione di GUS. </li></ol> 3. MUG saggio fluorimetrico: 8 ° giorno <ol><li> Isolare proteine ​​totali dalla macinazione di dischi foglia infiltrato in un mortaio con azoto liquido. Aggiungere 100 uL di tampone GUS estrazione (50 mM Napi pH 7,0, 10 mM EDTA, 0.1% Triton X-100, 0,1% N-lauroylsarcosine sale di sodio, β-mercaptoetanolo (0,7 microlitri / ml) mantenendo il campione in ghiaccio. </li><li> Centrifugare la sospensione a 14.000 g per 5 minuti a 4 ° C e prendere surnatante come totale delle proteine ​​solubili. </li><li> Misurare la concentrazione utilizzando un NanoDrop secondo le istruzioni del produttore. </li><li> Regolare la concentrazione di proteine ​​a 100 mg di proteina solubile totale per ogni campione mediante tampone di estrazione GUS. </li><li> Aggiungere substrato MUG (4-metilumbelliferil-β-D-glucuronide triidrato), preparato in tampone di estrazione GUS ad una concentrazione finale di 2 mm. Un volume totale di 100 l per ogni campione (proteine ​​+ substrato MUG) dovrebbe essere sufficiente. </li><li> Incubare le reazioni per 1 ora a 37 ° C. </li><li> Fermare le reazioni con l&#39;aggiunta di 800 ml di GUS fermare tampone (0,2 M Na 2 CO 3). </li><li> Misurare la fluorescenza utilizzando un lettore di piastre a una lunghezza d&#39;onda di eccitazione di 365 nm e lunghezza d&#39;onda di emissione di 455 nm e confrontare i valori di una curva MU standard. Livelli di relazione sulle attività GUS come MU nmol / mg proteina solubile / min. </li></ol> 4. Azioni e soluzioni: <ol><li> Acetosyringone (1 M) stock (FW = 196,2 g) </sub&gt; - 0,981 g in 5 ml di DMSO </li><li> Kanamicina (100 mg / ml) magazzino - preparare nel dH 2 0 - Filtro sterilizzare </li><li> Rifampicina (25 mg / ml) magazzino - la preparazione in DMSO </li><li> Luria-Bertani (LB) liquido terreni di coltura: (1L) 1% (w / v) bacto-triptone, 10 g 0,5% (w / v) bacto-estratto di lievito, 5 g 170 mM di cloruro di sodio 10 g pH - 7 </li><li> Acetosyringone (25 mM) = 25 ml di uno stock 1 M in un volume finale di 1 ml </li><li> Infiltrazione Medio: (1L) 10 mM MgSO 4 0,7 dH 2 0 (FW = 246,48 g), 2,4648 g 9 mm MES (FW = 213,25 g), 1,91925 g pH - 5.6 </li><li> Acetosyringone (100 mM) = 100 ml di uno stock 1M in un volume finale di 1 ml </li><li> GUS Tampone di estrazione Napi 50 mM pH 7,0, 10 mM EDTA, 0.1% Triton X-100, 0,1% N-lauroylsarcosine sale di sodio, β-mercaptoetanolo (0,7 microlitri / ml) </li><li> 2x Tampone fosfato 0,2 M NaH 2 PO 4 e 0,2 M Na 2 HPO 4 pH 7 </li><li> X-Gluc substrato Sciogliere 1 mg di 5-bromo-4-cloro-3-indolil BD-glucuronide (X-Gluc) in 0,1 ml di metanolo. Aggiungere 1 ml di tampone fosfato 2x, 20 microlitri 0,1 M ferricianuro di potassio, 10μl Triton X-100 10% e 850 ml di acqua distillata. </li></ol> 5. Rappresentante dei risultati: A seguito di questo protocollo, la perdita di espressione GUS è previsto quando si verifica la morte delle cellule. Abbiamo contemporaneamente espresso il gene reporter, β-glucuronidasi (GUS) con un membro del cito-protettiva Arabidopsis Bcl-2 associati athanoGene (UFSP) di famiglia. Siamo co-infiltrato N. benthamiana lascia con una cassetta 35S guidato espressione BORSA e il GUS vettore pCAMBIA 2301 utilizzando LBA4404 ceppo tumefaciens. Come mostrato nella figura 2, un aumento visibile nella colorazione GUS è stata osservata dopo questa infiltrazione. Al contrario, quando il noto pro-apoptotici membro della famiglia Bcl-2 BAX è stato utilizzato, una marcata riduzione della colorazione GUS è stata osservata (Fig. 2). In entrambi i casi, espressione di GUS era visibilmente diverso rispetto al controllo. Tuttavia, quando la differenza di espressione è meno evidente, saggi MUG fluorimetrico possono essere eseguite per quanitate espressione GUS. <img src="/files/ftp_upload/2680/2680fig1.jpg" alt="Figura 1"/> Figura 1. Esempio di infiltrazione miscela Agrobacterium del lato abaxial di N. benthamiana lascia con un 1 ml ago-meno siringa. <img src="/files/ftp_upload/2680/2680fig2.jpg" alt="Figura 2"/> Figura 2. Un vettore GUS è stato co-espresso in N. benthamiana lascia con la morte anti-gene, e un noto induttore di morte cellulare. Livelli GUS sono stati confrontati con un controllo vettore vuoto (GUS controllo).

<ol>
	<li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUS+fusions:+,+&#946;-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">GUS fusions: , &#946;-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3901 (1987).</li><li>Nishihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+&#946;-Glucuronidase+Gene+in+Pollen+of+Lily+(Lilium+longiflorum),+Tobacco+(Nicotiana+tabacum),+Nicotiana+rustica,+and+Peony+(Paeonia+lactiflora)+by+Particle+Bombardment.">Expression of the &#946;-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.</a> Plant Physiol. 102, 357-357 (1993).</li><li>Hodal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 87, 115-115 (1992).</li><li>Kabbage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BAG+proteins:+a+ubiquitous+family+of+chaperone+regulators.">The BAG proteins: a ubiquitous family of chaperone regulators.</a> Cell Mol. Life Sci. . 65, 1390-1390 (2008).</li><li>Kang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtBAG6,+a+novel+calmodulin-binding+protein,+induces+programmed+cell+death+in+yeast+and+plants.">AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.</a> Cell Death Differ. 13 (1), 84-84 (2006).</li><li>Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 165 (1), 1-1 (2006).</li><li>Takayama, S., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+targeting+and+regulation+by+BAG+family+proteins.">Molecular chaperone targeting and regulation by BAG family proteins.</a> Nat Cell Biol. 3 (10), 237-237 (2001).</li><li>Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+analysis+of+BAG-1:+a+novel+Bcl-2-binding+protein+with+anti-cell+death+activity.">Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity.</a> Cell. 80 (2), 279-279 (1995).</li><li>Vitha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+&#946;-glucuronidase+assay+in+transgenic+plants.">Quantitative &#946;-glucuronidase assay in transgenic plants.</a> Biol. Plant. 35, 151-151 (1993).</li><li>Otha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+Expression+in+Tobacco+of+a+&#946;-Glucuronidase+(GUS)+Reporter+Gene+Containing+an+Intron+Within+the+Coding+Sequence.">Construction and Expression in Tobacco of a &#946;-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence.</a> Plant Cell Physiol. 31, 805-805 (1990).</li></ol>

Nessun conflitto di interessi dichiarati.

<p class="jove_content"&gt; E &#39;spesso difficile da usare tecniche di individuazione delle cellule morte in impianti che sono comuni nei sistemi di mammiferi. In combinazione con un sistema di giornalista GUS, presentiamo una base vegetale, metodo sensibile per la rilevazione e l&#39;analisi di giocatori di morte cellulare. Questo metodo sfrutta il semplice fatto che le cellule vive sono necessari per l&#39;espressione GUS a verificarsi. Per garantire risultati significativi e la ripetibilità, è fondamentale che le culture ospitare la cassetta GUS e il gene per essere analizzati sono infiltrati in rapporti uguali. Questi rapporti devono essere mantenuti quando si confrontano ulteriori potenziali giocatori morte cellulare. Come mostrato sopra, abbiamo utilizzato con successo questo metodo nel nostro laboratorio per studiare la funzione potenziale morte di molti geni. A causa della sua semplicità, crediamo che questo sistema può essere utilizzato di routine per rilevare modulatori morte. Inoltre, il metodo può essere regolata per ospitare più geni in miscele singola cultura. Come a volte è il caso, la funzione di morte delle cellule di un particolare gene è condizionato alla sua attivazione / repressione da parte delle cellule di altri effettori morte programmata. Pertanto, geni multipli possono essere co-espressa in una miscela singola cultura con una cassetta GUS per indagare queste possibilità.</p&gt;

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Rifampicina </td><td> VWR </td><td> IC19549001 </td><td> Magazzino 25mg/ml in DMSO </td></tr><tr><td> 4&#39;-idrossi-3 &#39;, 5&#39;-dimethoxyacetophenone (Acetosyringone) </td><td> VWR </td><td> TCD2666 </td><td> 0,981 g / mL in DMSO (1M stock) </td></tr><tr><td> 2 - (4-morfolino) etano monoidrato (MES) </td><td> VWR </td><td> EM-6110 </td><td> 1,92 g / L in acqua per fare 1 litro di media infiltrazione </td></tr><tr><td> Bacto-triptone </td><td> Pescatore </td><td> BP1421-2 </td><td> 10 g / L in acqua per fare 1 l di media LB </td></tr><tr><td> Bacto-estratto di lievito </td><td> VWR </td><td> EM1.03753.0500 </td><td> 5 g / L in acqua per fare 1 l di media LB </td></tr><tr><td> Cloruro di sodio </td><td> VWR </td><td> EM-7710 </td><td> 10 g / L in acqua per fare 1 l di media LB </td></tr><tr><td> Sodio fosfato monobasico monoidrato </td><td> VWR </td><td> MK-7868-12 </td><td> 2.5g / L in acqua per fare 50 mM di tampone Napi </td></tr><tr><td> Sodio fosfato bibasico eptaidrato </td><td> VWR </td><td> EMD-SX0715-1 </td><td> 5 g / L in acqua per fare 50 mM di tampone Napi </td></tr><tr><td> Solfato di magnesio eptaidrato </td><td> VWR </td><td> EM-MX0070-1 </td><td> 2,5 g / L in acqua per fare 1 litro di media infiltrazione </td></tr><tr><td> N-Lauroylsarcosine </td><td> VWR </td><td> TCL0151-500G </td><td> 0,1% v / v in tampone GUS </td></tr><tr><td> 5-Bromo-4-cloro-3-indossile-beta-D-glucuronide sale cyclohexylammonium (X-Gluc) </td><td> Oro Biotecnologie </td><td> G1281C </td><td> 1mg/100uL in metanolo 100% </td></tr><tr><td> 4-metilumbelliferil-μ-D-glucuronide idrato (MUG) </td><td> Sigma </td><td> M5664 </td><td> 2 mM in 100 uL di tampone di estrazione GUS </td></tr><tr><td> Ferrocianuro di potassio triidrato </td><td> VWR </td><td> EM-PX1460-1 </td><td> Magazzino 100mM per X-Gluc soluzione substrato </td></tr><tr><td> β-Methylumbelliferone (MU) </td><td> Sigma-Aldrich </td><td> M1381 </td><td> Per MU standard di utilizzo del buffer di estrazione curva GUS </td></tr><tr><td> Carbonato di sodio </td><td> VWR </td><td> EM-SX0395-11 </td><td> 0,2 M in acqua per fare GUS tampone arresto </td></tr></tbody></table>

a &#946;-glucuronidase (gus) based cell death assay

Abbiamo sviluppato un nuovo sistema di espressione transiente pianta che esprime allo stesso tempo il gene reporter β-glucuronidasi (GUS), con putativi regolatori positivi o negativi di morte cellulare. In questo sistema, N. lascia benthamiana sono co-infiltrati con una cassetta 35S espressione guidato contenente il gene da analizzare, e il GUS vettore pCAMBIA 2301 utilizzando LBA4404 ceppo Agrobacterium come veicolo. Poiché le cellule vive sono necessari per l&#39;espressione GUS a verificarsi, perdita di attività GUS è previsto quando questo gene marcatore è co-espresso con regolatori positivi di morte cellulare. Allo stesso modo, un aumento dell&#39;attività GUS si osserva quando i geni anti-apoptotici vengono utilizzati rispetto al controllo vettoriale. Come mostrato di seguito, abbiamo utilizzato con successo questo sistema nel nostro laboratorio per analizzare entrambi i giocatori pro-e anti-morte. Questi includono l&#39;impianto anti-apoptotica Bcl-2 associati athanoGene (UFSP), la famiglia, così come, gli induttori conosciuti mammiferi di morte cellulare, come BAX. Inoltre, abbiamo utilizzato questo sistema per analizzare la funzione morte di troncamenti specifiche all&#39;interno di proteine, che potrebbe fornire indizi sulla possibile modificazione post-traduzionale / attivazione di queste proteine. Qui, vi presentiamo un metodo rapido e sensibile impianto base, come un primo passo nello studio delle funzioni morte di specifici geni.

Watch this Scientific Journal Video about A Î²-glucuronidase GUS Based Cell Death Assay at JoVE.com

A &#946;-glucuronidase GUS Based Cell Death Assay

Morte cellulare programmata test comunemente usati nei sistemi di mammiferi, come laddering DNA o saggi TUNEL, sono spesso difficili da riprodurre nelle piante. In combinazione con un sistema di giornalista GUS, proponiamo un rapido test basato impianti transitori per analizzare le proprietà morte potenziale di specifici geni.

Un β-glucuronidasi (GUS) Sulla base di analisi morte cellulare

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

a-glucuronidase-gus-based-cell-death-assay

Nanyang Technological University

Research

JoVE Journal

Biology

A &#946;-glucuronidase (GUS) Based Cell Death Assay

28.0K Views.  Texas A&M University. Morte cellulare programmata test comunemente usati nei sistemi di mammiferi, come laddering DNA o saggi TUNEL, sono spesso difficili da riprodurre nelle piante. In combinazione con un sistema di giornalista GUS, proponiamo un rapido test basato impianti transitori per analizzare le proprietà morte potenziale di specifici geni.

Video: A β-glucuronidase GUS Based Cell Death Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Studiare Biogenesi della membrana con una luciferasi saggio basato su Reporter Gene

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Ricerca

Biologia

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Biomolecolare fluorescenza complementazione (BiFC) Assay per interazione proteina-proteina nelle cellule Cipolla Utilizzando la Helios Gun Gene

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Modificato annessina V / propidio ioduro Apoptosis analisi di valutazione accurata di morte cellulare

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Modificato il mouse sulle cellule staminali Saggio base per quantificare l&#39;efficienza di induzione cardiogeno

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Saggio di immunoprecipitazione della cromatina per geni tessuto-specifici utilizzando nella fase iniziale di embrioni di topo

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

L&#39;uso di LysoTracker per rilevare la morte cellulare programmata in embrioni e differenziazione delle cellule staminali embrionali

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

Un'analisi del Reporter di fluorescenza-basata semplice per identificare i componenti cellulari necessari per tossina ricina una catena (RTA) tratta di lievito

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

Il test basato sulla microscopia per studiare e analizzare gli endosomi di riciclaggio utilizzando il traffico di SNARE

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Un metodo passo-passo per rilevare gli anticorpi neutralizzanti contro l'AAV utilizzando un test colorimetrico basato su cellule

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Un β-glucuronidasi (GUS) Sulla base di analisi morte cellulare

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