Name: cellular redox profiling using high-content microscopy
Uploaded: 2017-05-14T13:00:00
Description: Watch this Scientific Journal Video about Cellular Redox Profiling Using High-content Microscopy at JoVE.com

This research was supported by the University of Antwerp (TTBOF/29267, TTBOF/30112), the Special Research Fund of Ghent University (project BOF/11267/09), NB-Photonics (Project code 01-MR0110) and the CSBR (Centers for Systems Biology Research) initiative from the Netherlands Organization for Scientific Research (NWO; No: CSBR09/013V). Parts of this manuscript have been adapted from another publication1, with permission of Springer. The authors thank Geert Meesen for his help with the widefield microscope.

Il protocollo sottostante è descritto come eseguito per le cellule NHDF e con l&#39;uso delle piastre multivibranti specificate nel file dei materiali. Vedere la Figura 1 per una panoramica generale del flusso di lavoro. 1. Preparazione dei reagenti <ol><li> Preparare il mezzo completo integrando Dulbecco&#39;s Modified Eagle Medium (DMEM) con 10% v / v Fetal Bovine Serum (FBS) e 100 IU / mL penicillina e 100 UI / mL streptomicina (PS). Per 500 ml di media co...

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Questo documento descrive un metodo di microscopia ad alto contenuto per la quantificazione simultanea dei livelli ROS intracellulari e morfofunzione mitocondriale in NHDF. La sua performance è stata dimostrata con un caso di studio sul NHDF trattato con SQV. I risultati supportano evidenze precedenti dalla letteratura in cui sono stati osservati livelli aumentati di ROS o disfunzione mitocondriale dopo il trattamento con inibitori della proteasi HIV di tipo 1, anche se in esperimenti separati 19</sup...

<html> La concentrazione di ROS intracellulare è regolata minuziosamente attraverso una interazione dinamica tra i sistemi ROS e ROS. Lo squilibrio tra i due provoca uno stato di stress ossidativo. Tra le principali fonti di ROS sono i mitocondri 1 . Considerato il loro ruolo nella respirazione cellulare, sono responsabili della maggior parte delle molecole di superossido intracellulare (O 2 - - ) 2 . Ciò è dovuto principalmente alla perdita di elettroni a O 2 al complesso 1 della catena di trasporto di elettroni in condizioni di forte potenziale di membrana mitocondriale negativo (Δψ m ), vale a dire l&#39; iperpolarizzazione mitocondriale. D&#39;altra parte, la depolarizzazione mitocondriale è stata correlata anche con l&#39;aumento della produzione di ROS che punta a più modi di azione 3 , 4 , 5 ,&gt; 6 , 7 , 8 . Inoltre, attraverso modifiche di redox nelle proteine ​​delle macchine di fusione di fissione, ROS co-regola la morfologia mitocondriale 9. Ad esempio, la frammentazione è correlata all&#39;aumento della produzione ROS e all&#39;apoptosi 10 , 11 , mentre i mitocondri filamentosi sono stati legati alla fame nutritiva e alla protezione contro Mitophagy 12 . Data l&#39;intricata relazione tra ROS cellulare e morfofunzione mitocondriale, entrambi dovrebbero essere idealmente quantificati contemporaneamente nelle cellule viventi. Per fare esattamente questo, è stato sviluppato un saggio di imaging ad alto contenuto basato sulla microscopia a larga scala automatizzata e sull&#39;analisi delle immagini delle colture cellulari aderenti colorate con le sonde fluorescenti CM-H 2 DCFDA (ROS) e TMRM (mitocondriali Δψ m e morfologia). L&#39;imaging ad alto contenuto si riferisce all&#39;estrazione di spAtiemodally ricchi ( cioè , un gran numero di funzioni descrittive) informazioni sui fenotipi cellulari utilizzando marcatori complementari multipli e analisi automatiche delle immagini. In combinazione con la microscopia automatizzata molti campioni possono essere proiettati in parallelo ( cioè ad alta velocità) aumentando così la potenza statistica del dosaggio. Infatti, un elemento principale del protocollo è che consente la simultanea quantificazione di più parametri nella stessa cella, e questo per un gran numero di celle e condizioni. Il protocollo è suddiviso in 8 parti (descritte in dettaglio nel protocollo sottostante): 1) Celle di semina in una piastra a 96 pozzetti; 2) Preparazione di soluzioni di magazzino, soluzioni di lavoro e buffer di imaging; 3) Impostazione del microscopio; 4) Caricamento delle cellule con CM-H 2 DCFDA e TMRM; 5) Primo biografo live per misurare i livelli basali ROS e la morfofunzione mitocondriale; 6) Secondo round di immagini in diretta dopo l&#39;aggiunta di tert- butilePerossido (TBHP) per misurare i livelli di ROS indotti; 7) Analisi automatica delle immagini; 8) Analisi dei dati, controllo della qualità e visualizzazione. Il dosaggio è stato originariamente sviluppato per i normali fibroblasti umani umani (NHDF). Poiché queste celle sono grandi e piatte, sono adatte per la valutazione della morfologia mitocondriale nelle immagini 2D widefield 13 , 14 . Tuttavia, con modifiche minori, questo metodo è applicabile ad altri tipi di cellule aderenti. Inoltre, accanto alla combinazione di CM-H 2 DCFDA e TMRM, il flusso di lavoro è conforme a una varietà di coppie di coloranti fluorescenti con diverse specificità molecolari 1 , 15 .

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM)</td>
			<td>ThermoFisher Scientific</td>
			<td>T668</td>
			<td></td>
		</tr>
		<tr>
			<td>CM-H2DCFDA (General Oxidative Stress Indicator)</td>
			<td>ThermoFisher Scientific</td>
			<td>C6827</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma)Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>MatriPlate 96-Well Glass Bottom MicroWell Plate 630 &#181;L-Black 0.17 mm Low Glass Lidded</td>
			<td>Brooks life science systems</td>
			<td>MGB096-1-2-LG-L</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS w/o Phenol Red 500 ml</td>
			<td>Lonza</td>
			<td>BE10-527F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose with L-glutamine</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Bufered Saline (PBS) w/o Ca and Mg</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M 500mL</td>
			<td>Lonza</td>
			<td>17-737F</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene (EDTA) Solution</td>
			<td>Lonza</td>
			<td>BE17-161E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure F(ab&#39;)&#8322; Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson</td>
			<td>711-166-152</td>
			<td>Antibody used for acquiring flat-field image</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure F(ab&#39;)&#8322; Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson</td>
			<td>711-546-152</td>
			<td>Antibody used for acquiring flat-field image</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti eclipse widefield microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfect Focus System (PFS)</td>
			<td>Nikon</td>
			<td></td>
			<td>hardware-based autofocus system</td>
		</tr>
		<tr>
			<td>CFI Plan Apo Lambda 20x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elelements Advanced Research 4.5 with JOBS module</td>
			<td>Nikon</td>
			<td></td>
			<td>This software is used to steer the microscope and program/perform the automatic image acquisition prototocol</td>
		</tr>
		<tr>
			<td>ImageJ (FIJI) Version 2.0.0-rc-43/1.50g</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RStudio Version 1.0.44</td>
			<td>Rstudio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>R version 3.3.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cellular redox profiling using high-content microscopy

Le specie reattive di ossigeno (ROS) regolano i processi cellulari essenziali, inclusi l&#39;espressione genica, la migrazione, la differenziazione e la proliferazione. Tuttavia, livelli eccessivi di ROS inducono uno stato di stress ossidativo, accompagnato da danni ossidativi irreversibili al DNA, ai lipidi e alle proteine. Quindi, la quantificazione di ROS fornisce un proxy diretto per la condizione di salute cellulare. Poiché i mitocondri sono tra le principali fonti cellulari e gli obiettivi della ROS, l&#39;analisi congiunta della funzione mitocondriale e della produzione ROS nelle stesse cellule è fondamentale per una migliore comprensione dell&#39;interconnessione in condizioni fisiopatologiche. Pertanto, è stata sviluppata una strategia basata su microscopia ad alto contenuto per la quantificazione simultanea dei livelli ROS intracellulari, del potenziale della membrana mitocondriale (ΔΨ m ) e della morfologia mitocondriale. Si basa su microscopia automatizzata a fluorescenza a larga scala e analisi delle immagini di cellule adesive viventi, coltivate in piastre a più pozzetti e staineD con le cellule-permeabili fluorescenti reporter molecole CM-H 2 DCFDA (ROS) e TMRM (ΔΨ m e morfologia mitocondriale). In contrasto con la fluorimetria o la cytometria a flusso, questa strategia consente di quantificare i parametri subcellulari a livello della singola cellula con elevata risoluzione spaziale, sia prima che dopo la stimolazione sperimentale. Importante, la natura basata sull&#39;immagine del metodo consente di estrarre parametri morfologici oltre alle intensità del segnale. Il set di funzioni combinato viene utilizzato per l&#39;analisi dei dati multivariata esplorativa e statistica per rilevare le differenze tra sottopopolazioni, tipi di cellule e / o trattamenti. Qui viene fornita una descrizione dettagliata del dosaggio, insieme ad un esperimento di esempio che dimostra il suo potenziale per una discriminazione inequivocabile tra stati cellulari dopo la perturbazione chimica.

Il dosaggio è stato valutato utilizzando diversi esperimenti di controllo, i cui risultati sono descritti in Sieprath et al. 1 . In breve, la risposta di fluorescenza di CM-H 2 DCFDA e TMRM a variazioni indotte in modo estraneo nei ROS intracellulari e in ψ m, è stata quantificata per determinare la gamma dinamica. Per CM-H 2 DCFDA, NHDF ha mostrato un aumento lineare nel segnale di fluorescenza quando trattato con conc...

Watch this Scientific Journal Video about Cellular Redox Profiling Using High-content Microscopy at JoVE.com

Cellular Redox Profiling Using High-content Microscopy

Questo documento presenta un flusso di lavoro di microscopia ad alto contenuto per la quantificazione simultanea dei livelli ROS intracellulari, nonché il potenziale e la morfologia della membrana mitocondriale - definiti congiuntamente come morfofunzione mitocondriale - nelle cellule adesive viventi utilizzando le molecole reporter fluorescenti per cellule permeanti 5- 6) clorometil-2 &#39;, 7&#39;-diclorodidro-fluoresceina diacetato, acetil estere (CM-H 2 DCFDA) e tetrametilrodamina metilestere (TMRM).

Profilatura cellulare Redox usando la microscopia ad alto contenuto

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

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