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 cass...

<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 &amp;#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

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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 &#946;-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 ...

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Biologia

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 ...

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 ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Nozioni di base sull'istologia e rilevamento della morte cellulare nel tessuto delle api

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

Estensione della durata della vita dei neutrofili con CLON-G e un saggio di morte spontanea in vitro

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

Quantificazione dell'inizio della morte cellulare attraverso l'imaging continuo

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

Autore in primo piano: Un approccio semplificato allo studio dell'inizio della morte cellulare nella risposta ipersensibile 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Studiare l'inizio della morte cellulare utilizzando un microscopio digitale

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...