Questo studio è stato supportato da una scienze naturali e ingegneria del Canada (NSERC) e un assegno di ricerca Alberta Agricoltura Finanziamento Consorzio concedere al DRB. AMR è supportata tramite una borsa di studio Vanier NSERC canadese Graduate, una Università di assistentato insegnamento Alberta e la regina Elisabetta II borsa di studio universitari.

Questa procedura può essere eseguita in 500 microlitri tubi siliconato o 6 ml in polistirolo a fondo rotondo tubi FACS. Quest&#39;ultimo è normalmente utilizzati nello svolgimento di analisi di fattibilità in collaborazione con altre procedure. Tutti i volumi indicati sono per 6 ml di polistirolo a fondo rotondo tubi FACS. Per 500 microlitri tubi di silicone e ridurre tutti i volumi da 1 / 5. La concentrazione ottimale per l&#39;analisi di citometria a flusso è di 2-4 x 10 6 cellule per volume di 200 microlitri. Perdita di cellule possono derivare da questa procedura, e quindi si consiglia di ogni campione è composto da 4 x 10 6 cellule all&#39;inizio della procedura. 1. Preparazione delle cellule <ol><li> Celle di raccolta - si riferiscono a procedure specifiche per le linee di cellule corrispondente o isolamenti cellula primaria. </li><li> Centrifugare i campioni a 335 x g per 10 minuti e decantare il surnatante. </li><li> Risospendere le cellule in 2 ml 1 x soluzione salina tamponata con fosfato (PBS) - / - (senza calcio, senza magnesio). </li><li> Centrifugare i campioni a 335 x g per 10 minuti e decantare il surnatante. </li><li> Risospendere le cellule in 1 ml di buffer di 1 x annessina V vincolanti. </li><li> Centrifugare i campioni a 335 x g per 10 minuti e decantare il surnatante. </li><li> Risospendere le cellule in 100 ul buffer di 1 x annessina V vincolanti. </li></ol> 2. Applicazione di annessina V / PI Stain <ol><li> Aggiungi annessina V in base alle raccomandazioni del fabbricante [ad esempio 5 microlitri annessina V Alexa Fluor 488 (Molecular Probes, A13201)]. </li><li> Incubare le provette al buio per 15 minuti a temperatura ambiente. </li><li> Aggiungere 100 ml di buffer di 1 x annessina V vincolante per ciascun tubo di reazione. Ci dovrebbe essere di circa 200 l in ciascun tubo. </li><li> Aggiungere 4 ml di PI (Sigma, Cat # P-4864-10ML) che è stato diluito 1:10 in 1 tampone x annessina V vincolante (cioè 1 PI microlitri con 9 microlitri buffer di 1 x annessina V vincolante). Questo produrrà una concentrazione finale PI di 2 mg / ml in ciascun campione. </li><li> Incubare le provette al buio per 15 minuti a temperatura ambiente. </li><li> Aggiungere 500 microlitri di buffer 1 x annessina V vincolante per lavare le cellule. </li><li> Centrifugare i campioni a 335 x g per 10 minuti e decantare il surnatante. </li><li> Risospendere le cellule in 500 microlitri buffer di 1 x annessina V vincolante e 500 microlitri di formaldeide al 2% per creare una soluzione all&#39;1% di formaldeide (fissativo). Mescolare tubi da sfogliando gentile. </li><li> Fissare i campioni in ghiaccio per 10 minuti. In alternativa, i campioni possono essere conservati notte a 4 ° C al buio. Se il metodo di quest&#39;ultimo è scelto, assicuratevi di essere coerente in tutti i tubi etichettati con annessina V / PI (compresi i controlli di compensazione). </li><li> Aggiungere 1 ml 1 x PBS - / - per ogni campione e miscelare delicatamente sfogliando. </li><li> Centrifugare i tubi a 425 x g per otto minuti e decantare il surnatante. </li><li> Ripetere i passaggi 2.10 e 2.11. </li><li> Risospendere le sfere muovendo il tubo. </li><li> Aggiungere 16 ml di diluito 1:100 RNAsi A (Sigma, R4642) per ottenere una concentrazione finale di 50 mg / ml. Incubare per 15 minuti a 37 ° C. </li><li> Aggiungere 1 ml 1 x PBS - / - e mescolare delicatamente dal sfogliando. </li><li> Centrifugare i tubi a 425 x g per otto minuti. </li><li> I campioni sono ora pronti per essere analizzati. In alternativa, i campioni possono essere utilizzati per la colorazione passi successivi se annessina / PI colorazione viene eseguita in parallelo con altre procedure. </li></ol> 3. Rappresentante dei risultati: Esempi di tipi di cellule e linee cellulari che presentano diversi gradi di falsi positivi colorazione PI sono mostrati in Figura 1. Per spiegare il falso-positivi gli eventi, le cellule sono state fissate e poi trattati con RNasi A. Questo si traduce in una diminuzione significativa del numero di falsi positivi eventi, rispetto alle cellule che non siano sottoposti a trattamento RNasi (Figura 2B). Figura 2C mostra immagini rappresentative di cellule che sottoposti a trattamento RNAsi, rispetto alle cellule che non ha avuto un trattamento RNAsi. La Figura 3 mostra come la modifica annessina V / PI protocollo, con la fissazione e fasi di trattamento RNase, migliora protocolli convenzionali, limitando il numero di falsi positivi eventi di colorazione. <img src="/files/ftp_upload/2597/2597fig1.jpg" alt="Figura 1" /> Figura 1. Protocolli di colorazione convenzionale propidio ioduro di portare ad un significativo numero di falsi eventi positivi. Abbiamo già valutato estensione della colorazione falsa positiva attraverso le cellule primarie attraverso una vasta gamma di modelli animali e in una varietà di linee cellulari 10. Immagini rappresentative mostrano estensione della colorazione falsa positiva in tre popolazioni di cellule unico (una linea cellulare di uso comune - macrofagi RAW e due cellule primarie - murino macrofagi del midollo osseo (BMM) e macrofagi reni pesci rossi primaria (PKM)). La vera eventi positivi visualizzare l&#39;atteso co-localizzazione tra PI e DRAQ5 all&#39;interno del compartimento nucleare (aree giallo raffigurano colocalizzazione tra PI e DRAQ5). DRAQ5 è altamente membranene permeabile colorante che con precisione le macchie vano nucleare in cellule vive e morte 10,11,12. Per facilitare la determinazione visiva della colocalizzazione DRAQ5 macchia è stata data una pseudocolour verde. <img src="/files/ftp_upload/2597/2597fig2.jpg" alt="Figura 2" /> Figura 2. Migliorare l&#39;accuratezza della colorazione PI nucleare. (A) hanno valutato l&#39;accuratezza della colorazione PI nucleare basato sul grado di similarità di una serie di ben caratterizzato macchie nucleare (BrdU, 7-AAD e DAPI) per DRAQ5. BrdU è un nucleoside sintetico che viene incorporato nel DNA delle cellule proliferanti, e come tale sarà unica macchia all&#39;interno del compartimento nucleare. 7-AAD ha un&#39;alta affinità per il DNA e, simile a PI, non può attraversare una membrana plasmatica intatto. DAPI ha anche una forte affinità per il DNA nucleare ed è il più comunemente usato macchia nella microscopia a fluorescenza. Il grado di somiglianza era&gt; 99% tra BrdU, 7-AAD, DAPI e DRAQ5, evidenziando la loro capacità di macchiare con precisione il nucleo della cellula in macrofagi RAW 264.7. Al contrario, la colorazione citoplasmatica PI sulla base di protocolli convenzionali porta ad una diminuzione marcata somiglianza per cento della colorazione rispetto al DRAQ5. (B) Risultati simili sono stati osservati in due cellule primarie testate: murino macrofagi del midollo osseo (BMM) e macrofagi reni pesci rossi primaria (PKM). L&#39;incorporazione di 50 mg / ml RNasi A in fase di specifici all&#39;interno della procedura di colorazione rimuove non nucleari colorazione PI e aumenta in modo significativo il grado di somiglianza rispetto alla DRAQ5 colorazione. (C) immagini Rappresentante dei macrofagi renali primari mostrano il grado di somiglianza per celle con e senza trattamento RNAsi. Per facilitare la determinazione visiva delle differenze tra i trattamenti, DRAQ5 è stata data una verde. Aree giallo raffigurano colocalizzazione tra BrdU e DRAQ5 e PI e DRAQ5. <img src="/files/ftp_upload/2597/2597fig3.jpg" alt="Figura 3" /> Figura 3. Modificato annessina V / PI riduce significativamente l&#39;apoptosi false / eventi necrotico. Macrofagi primari reni pesci rossi (PKM) sono state colorate con convenzionali e modificato annessina V / PI protocolli. Scatterplot raffigurano l&#39;annessina V / PI macchia in PKM pesci rossi. La dispersione a sinistra mostra convenzionale annessina V / PI colorazione (non RNasi) di cellule PKM. La dispersione a destra mostra PKM cellule colorate con il annessina V modificato / PI protocollo (con RNasi). L&#39;aggiunta di risultati RNase in una marcata riduzione della colorazione PI a causa della rimozione di macchie di falsi positivi. PKM cellule sono state poi analizzate in base a distinte popolazioni all&#39;interno delle culture e l&#39;inizio progenitori (PE), monociti (Mono) e macrofagi maturi (Mat mac). La riduzione del numero di eventi PI positivo, dovuto alla rimozione del falso eventi positivi è più pronunciata nelle grandi cellule macrofagi maturi che in piccole cellule progenitrici presto. Conclusioni basate su protocolli convenzionali avrebbe erroneamente suggerito una correlazione positiva con la morte cellulare per i sottoinsiemi dei macrofagi più maturi.

<ol>
	<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J Immunol Methods. 184, 39-51 (1995).</li><li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+apoptotic+cell+death.">Flow cytometry of apoptotic cell death.</a> J Immunol Methods. 243, 167-190 (2000).</li><li>Cornelissen, M., Philippe, J., De Sitter, S., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes:+An+electron+microscopic+evaluation.">Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation.</a> Apoptosis. 7, 41-47 (2002).</li><li>Fried, J., Perez, A. G., Clarkson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytofluorometric+analysis+of+cell+cycle+distributions+using+propidium+iodide.+Properties+of+the+method+and+mathematical+analysis+of+the+data.">Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.</a> J Cell Biol. 71, 172-181 (1976).</li><li>Bacso, Z., Everson, R. B., Eliason, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+of+Annexin+V-binding+apoptotic+cells+is+highly+fragmented.">The DNA of Annexin V-binding apoptotic cells is highly fragmented.</a> Cancer Res. 60, 4623-4628 (2000).</li><li>Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P., Traganos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+apoptotic+cells+measured+by+flow+cytometry.">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13, 795-808 (1992).</li><li>Kroemer, G., Dallaporta, B., Resche-Rigon, 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+mitochondrial+death/life+regulator+in+apoptosis+and+necrosis.">The mitochondrial death/life regulator in apoptosis and necrosis.</a> Annu. Rev. Physiol. 60, 619-642 (1998).</li><li>Denecker, G., Vercammen, D., Declercq, W., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors.">Apoptotic and necrotic cell death induced by death domain receptors.</a> Cell Mol. Life Sci. 58, 356-370 (2001).</li><li>Faleiro, L., Lazebnik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+disrupt+the+nuclear-cytoplasmic+barrier.">Caspases disrupt the nuclear-cytoplasmic barrier.</a> J Cell Biol. 151, 951-959 (2000).</li><li>Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+apoptosis+assays+using+propidium+iodide+generate+a+significant+number+of+false+positives+that+prevent+accurate+assessment+of+cell+death.">Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.</a> J Immunol Methods. 358, 81-92 (2010).</li><li>Edward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Use+of+DNA-specific+anthraquinone+dyes+to+directly+reveal+cytoplasmic+and+nuclear+boundaries+in+live+and+fixed+cells.">Minireview: Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells.</a> Mol. Cells. 27, 391-396 (2009).</li><li>Njoh, K. L., Patterson, L. H., Zloh, M., Wiltshire, M., Fisher, J., Chappell, S., Ameer-Beg, S., Bai, Y., Matthews, D., Errington, R. J., Smith, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+analysis+of+the+DNA+targeting+bisalkylaminoanthraquinone+DRAQ5+in+intact+living+cells.">Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.</a> Cytometry Part A. 69, 805-814 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;annessina V / PI protocollo qui presentato è una versione modificata di protocolli convenzionali e tiene conto della presenza di RNA nel citoplasma che ha anche affinità per PI. Introduzione di RNase A (50 mg / ml) a seguito di un passo di formaldeide 1% fissazione ritardo nella procedura di colorazione migliora sensibilmente la precisione di colorazione nucleare PI. Effetti negativi sono stati osservati in colorazione nucleare PI o membrana plasmatica annessina V colorazione. Senza RNAsi Un trattamento, fino al 40% dei risultati PI macchia può portare a falsi eventi positivi, che porta ad un impatto potenzialmente significativo e negativo sulle conclusioni a valle 10. La nostra modificato annessina V / PI protocollo di colorazione è semplice ed è stato utilizzato in modo efficace in un&#39;ampia gamma di tipi di cellule (cellule primarie: murino macrofagi del midollo osseo e splenociti; suina cellule residenti del polmone, i macrofagi alveolari dei polmoni, isola dei linfonodi mesenterici, leucociti del sangue periferico , splenociti; cellule Blastoderm pollo; macrofagi reni pesci rossi primaria; leucociti del sangue periferico carpa; linee cellulari: RAW 264,7 macrofagi, le cellule T Jurkat, suina 3D4/31 macrofagi, CCL-71 fibroblasti pinna pesce rosso, pesce gatto 3B11 cellule B 10). E &#39;importante notare che le cellule sono colpite in modo differenziato da falsi colorazione PI positivo, con pile in genere hanno un maggior numero di falsi eventi positivi 10. La colorazione falsa PI differenze positive tra queste popolazioni cellulari può essere parzialmente attribuito a differenze di dimensioni delle cellule, ma può anche derivare da differenze nel contenuto di RNA. Come tale, l&#39;incorporazione di questa procedura è particolarmente rilevante per i sistemi sperimentali che utilizzano, tra gli altri, cellule che subiscono lo stress genotossico, cellule trattate con farmaci arresto del ciclo cellulare come la timidina o idrossiurea, cellule infettate infettati, e gli studi sulle cellule embrionali in cui la progressione evolutiva è caratterizzato da variazioni discrete cellulari sintesi dell&#39;RNA.

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<td>1 x PBS-/-</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>1 x Annexin V Binding Buffer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556454</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Molecular Probes (Invitrogen)</td>
<td>A13201</td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide (PI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
<td>1 mg/mL in H2O</td>
</tr>
<tr>
<td>2% Formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F1268</td>
<td>&nbsp;</td>
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<td>RNase A from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R4642</td>
<td>&nbsp;</td>
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<td>DRAQ5</td>
<td>&nbsp;</td>
<td>Biostatus</td>
<td>DR50050</td>
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modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death

Studi di apoptosi cellulare sono stati significativamente influenzati dall&#39;introduzione della citometria a flusso basato su metodi. Ioduro di propidio (PI) è ampiamente utilizzato in combinazione con annessina V per determinare se le cellule sono vitali, apoptotiche o necrotiche attraverso differenze di integrità della membrana plasmatica e 1,2 permeabilità. Il V / PI annessina protocollo è un metodo comunemente usato per lo studio delle cellule apoptotiche 3. PI è usato più spesso di altre macchie nucleare perché è economica, stabile e un buon indicatore della vitalità cellulare, basato sulla sua capacità di escludere la tintura in cellule viventi 4,5. La capacità di PI di inserire una cella dipende dalla permeabilità della membrana; PI non macchia cellule apoptotiche dal vivo o inizio a causa della presenza di una membrana plasmatica intatto 1,2,6. Alla fine di cellule apoptotiche e necrotiche, l&#39;integrità delle membrane plasmatiche e nucleari diminuisce 7,8, permettendo PI di passare attraverso le membrane, intercalate in acidi nucleici, e visualizzare fluorescenza rossa 1,2,9. Purtroppo, ci accorgiamo che convenzionale annessina V / PI protocolli di portare ad un significativo numero di falsi eventi positivi (fino al 40%), che sono associati con colorazione PI di RNA all&#39;interno del compartimento citoplasmatico 10. Cellule primarie e linee cellulari in una vasta gamma di modelli animali sono colpiti, con celle di grandi dimensioni (nucleare: i rapporti citoplasmatica &lt;0,5) che mostra la più alta presenza 10. Qui, si dimostra una versione modificata annessina V / PI metodo che fornisce un significativo miglioramento per la valutazione della morte cellulare rispetto ai metodi tradizionali. Questo protocollo si avvale delle variazioni di permeabilità cellulare durante la divisione cellulare di fissaggio per promuovere ingresso di RNase A nelle cellule dopo la colorazione. Sia i tempi e la concentrazione di RNase A sono state ottimizzate per la rimozione di RNA citoplasmatici. Il risultato è un miglioramento significativo rispetto ai tradizionali annessina V / PI protocolli (&lt;5% di eventi con colorazione citoplasmatica PI).

Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

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

Un metodo accurato per la valutazione della morte cellulare è descritto. Il protocollo migliora convenzionale annessina V / propidio ioduro (PI), i protocolli, che mostra fino al 40% di falsi positivi eventi in linee cellulari e cellule primarie da una vasta gamma di modelli animali.

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-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Research

JoVE Journal

Biology

102.6K Views.  University of Alberta. Un metodo accurato per la valutazione della morte cellulare è descritto. Il protocollo migliora convenzionale annessina V / propidio ioduro (PI), i protocolli, che mostra fino al 40% di falsi positivi eventi in linee cellulari e cellule primarie da una vasta gamma di modelli animali.

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

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 ...

Ricerca

Biologia

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

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

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

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

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 ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Attivazione di apoptosi da Microiniezione citoplasmatici del Citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

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 ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Il pomodoro / GFP-FLP / FRT Metodo per Live Imaging del Mosaico adulti<em&gt; Drosophila</em&gt; cellule fotorecettrici

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Strategie per l&#39;inseguimento Anastasis, una cellula di sopravvivenza fenomeno che Inverte Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Valutazione della Selective mRNA Traduzione in cellule di mammifero per polisoma Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Un saggio di comunicazione intercellulare Junction ioduro-giallo fluorescente proteina-Gap

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Test di coltura di una singola miofibra per la valutazione della funzionalità delle cellule staminali muscolari adulte Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

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 ...