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.

Protokollen nedenfor beskrives som udført for NHDF-celler og ved anvendelse af multiwellpladerne, der er angivet i materialefilen. Se Figur 1 for et generelt overblik over arbejdsgangen. 1. Fremstilling af reagenser <ol><li> Forbered komplet medium ved at supplere Dulbecco&#39;s Modified Eagle Medium (DMEM) med 10% v / v føtalt bovint serum (FBS) og 100 IE / ml penicillin og 100 IE / ml streptomycin (PS). Til 500 ml komplet medium tilsættes 50 ml FBS og 5 m...

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I dette papir beskrives en højindholdsmikroskopimetode til samtidig kvantificering af intracellulære ROS-niveauer og mitokondriel morfofunktion i NHDF. Dens præstation blev demonstreret ved en casestudie om SQV-behandlet NHDF. Resultaterne understøtter tidligere beviser fra litteratur, hvor der er observeret forhøjede ROS-niveauer eller mitokondrielle dysfunktion efter behandling med HIV-proteasehæmmere af type 1, om end i separate forsøg 19 , 20 ,</...

<html> Koncentrationen af ​​intracellulær ROS reguleres omhyggeligt gennem et dynamisk samspil mellem ROS-producerende og ROS-defusionssystemer. Ubalance mellem de to fremkalder en tilstand af oxidativ stress. Blandt de vigtigste kilder til ROS er mitokondrier 1 . På grund af deres rolle i cellulær respiration er de ansvarlige for størstedelen af ​​intracellulære superoxid (O 2 • - ) molekyler 2 . Dette skyldes for det meste elektrondækage til O2 i kompleks 1 af elektrontransportkæden under betingelser med stærkt negativt indre mitokondrialmembranpotentiale (ψψ m ), dvs. mitokondriel hyperpolarisering. På den anden side er mitokondrielt depolarisering også korreleret med øget ROS-produktion, der peger på flere virkemåder 3 , 4 , 5 ,&gt; 6 , 7 , 8 . Endvidere korrigerer ROS ved hjælp af redoxmodifikationer i proteiner fra fissionsfusionsmaskineriet mitokondriemorfologi 9. For eksempel er fragmentering korreleret med øget ROS-produktion og apoptose 10 , 11 , mens filamentøse mitokondrier er blevet forbundet med næringsstult og beskyttelse mod Mitofagi 12 . I betragtning af det indviklede forhold mellem cellulær ROS og mitokondrialt morfofunktion, bør begge ideelt set kvantificeres samtidigt i levende celler. For at gøre netop dette blev der udviklet et billeddannelsesassay med høj indhold baseret på automatiseret widefieldmikroskopi og billedanalyse af adherente cellekulturer farvet med fluorescerende prober CM-H 2 DCFDA (ROS) og TMRM (mitokondriel Δψ m og morfologi). Højindhold billeddannelse refererer til udvinding af spAtiotemporalt rige ( dvs. stort antal beskrivende egenskaber) information om cellulære fænotyper ved hjælp af flere komplementære markører og automatiserede billedanalyser. Når de kombineres med automatiseret mikroskopi, kan mange prøver screenes parallelt ( dvs. høj gennemstrømning) og derved øge analysens statistiske effekt. Faktisk er et primært aktiv i protokollen, at det giver mulighed for samtidig kvantificering af flere parametre i samme celle, og dette for et stort antal celler og betingelser. Protokollen er opdelt i 8 dele (beskrevet detaljeret i protokollen nedenfor): 1) Seedceller i en 96-brønds plade; 2) Fremstilling af stamopløsninger, arbejdsløsninger og billeddannelsesbuffer; 3) Opsætning af mikroskop; 4) Indlæsning af cellerne med CM-H2 DCFDA og TMRM; 5) Første levende billeddannelsesrunde til måling af basale ROS niveauer og mitokondriel morphofunktion; 6) Anden levende billeddannelsesrunde efter tilsætning af tert -butylPeroxid (TBHP) til måling af inducerede ROS-niveauer; 7) Automatiseret billedanalyse; 8) Dataanalyse, kvalitetskontrol og visualisering. Assayet blev oprindeligt udviklet til normale humane dermal fibroblaster (NHDF). Da disse celler er store og flade, er de velegnede til vurdering af mitokondriamorfologi i 2D widefield-billeder 13 , 14 . Men med mindre ændringer er denne metode gældende for andre vedhængende celletyper. Desuden er arbejdsflowet ved siden af ​​kombinationen af ​​CM-H 2 DCFDA og TMRM i overensstemmelse med en række fluorescerende farvestofpar med forskellige molekylære specificiteter 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

Reaktive oxygenarter (ROS) regulerer væsentlige cellulære processer, herunder genekspression, migration, differentiering og proliferation. Overdrivne ROS-niveauer fremkalder imidlertid en tilstand af oxidativ stress, som ledsages af irreversibel oxidativ skade på DNA, lipider og proteiner. Således giver kvantificering af ROS en direkte proxy for cellulær sundhedstilstand. Da mitokondrier er blandt de store cellulære kilder og mål for ROS, er fælles analyse af mitochondrielle funktion og ROS-produktion i de samme celler afgørende for bedre at forstå sammenkoblingen i patofysiologiske forhold. Derfor blev en højmikroskopibaseret strategi udviklet til samtidig kvantificering af intracellulære ROS-niveauer, mitokondrielt membranpotentiale (ΔΨ m ) og mitokondrielmorfologi. Den er baseret på automatiseret widefield fluorescensmikroskopi og billedanalyse af levende vedhæftende celler, der vokser i plader med flere brønde og staineD med de celle-permeable fluorescerende reportermolekyler CM-H2 DCFDA (ROS) og TMRM (ΔΨm og mitokondrielmorfologi). I modsætning til fluorimetri eller flowcytometri tillader denne strategi kvantificering af subcellulære parametre på niveauet af den enkelte celle med høj spatiotemporal opløsning, både før og efter forsøgsstimulering. Det er vigtigt, at den billedbaserede natur af metoden tillader ekstraktion af morfologiske parametre ud over signalintensiteter. Det kombinerede funktionssæt bruges til eksplorativ og statistisk multivariat dataanalyse til at detektere forskelle mellem subpopulationer, celletyper og / eller behandlinger. Her gives en detaljeret beskrivelse af analysen sammen med et eksempeleksperiment, der viser dets potentiale for entydig forskelsbehandling mellem cellulære tilstande efter kemisk forstyrrelse.

Assayet er blevet benchmarked ved anvendelse af adskillige kontrolforsøg, hvis resultater er beskrevet i Sieprath et al. 1 . Kort fortalt er fluorescensresponsen af ​​CM-H2 DCFDA og TMRM til extrant inducerede ændringer i intracellulær ROS og Δψm blevet kvantificeret til bestemmelse af det dynamiske område. For CM-H2 DCFDA viste NHDF en lineær stigning i fluorescenssignalet, når det blev behandlet med stigende koncentrationer af TBHP mellem et...

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

Cellular Redox Profiling Using High-content Microscopy

I dette papir præsenteres en mikroskopisk arbejdsstrøm med høj indhold til samtidig kvantificering af intracellulære ROS-niveauer samt mitokondrielt membranpotentiale og morfologi - i fællesskab betegnet mitokondriel morfofunktion - i levende adhærente celler ved anvendelse af de cellemeorante fluorescerende reportermolekyler 5- (og- 6) -chloromethyl-2 &#39;, 7&#39;-dichlorodihydrofluoresceindiacetat, acetylester (CM-H2 DCFDA) og tetramethylrhodaminmethylester (TMRM).

Cellular Redox Profiling ved hjælp af højindholdsmikroskopi

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

Research

JoVE Journal

Biology

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Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

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Glycan Profiling of Plant Cell Wall Polymers using Microarrays

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Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Et højt indhold Imaging Workflow til Uddannelse Grb2 signalering Komplekser ved ekspressionskloning

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Klonale Sporing af hæmatopoietisk stamcelletransplantation og progenitorceller Præget af Fem fluorescerende proteiner ved hjælp af konfokal og multiphoton mikroskopi

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Tandem Højtryks-Frysning og Quick Freeze Substitution af plantevævene for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

Høj Content Screening Analyse til at vurdere toksikologiske virkninger af skadelige og potentielt skadelige Bestanddele (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Open Source High Content Analysis hjælp Automated Fluorescens Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...