Name: optimization of the wound scratch assay to detect changes in murine mesenchymal stromal cell migration after damage by soluble cigarette smoke extract
Uploaded: 2015-12-03T10:15:00
Description: Watch this Scientific Journal Video about Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract at JoVE

We are grateful to the other members of the lab for their technical assistance in developing the CSE damage assay, and their support and advice during the development of this assay. In addition, we are grateful to the Holy Cross Fenwick Scholar Committee for their support of Nicholas Cormier, and the Holy Cross College Honors program for their support of Alexander Yeo.

Bemærk: Til denne undersøgelse, lunge mesenkymale stromaceller (LR-MSC&#39;er) fra den distale lungevæv blev isoleret enten baseret på deres ekspression af celleoverflademarkører (CD45 neg, CD31 neg, Sca-1 høj, EpCAM neg) 14,15, ved hjælp af eksplantat out-vækst 16, eller ved hjælp af enzymatisk fordøjelse 17. Adhærerende LR-MSC&#39;er dyrkedes i DMEM med høj glucose og ingen glutamin, 15% FBS, 1x antibiotisk / antimykotisk og 2 mM glutamin (herefter ful...

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Den her beskrevne protokol giver et kvantitativt robust, standardiseret metode til at udføre og analysere scratch analyser. Simple scratch analyser rutinemæssigt anvendes i mange forskellige områder af undersøgelsen for at undersøge cellulære migration. Men traditionelt scratch-analysen har manglet en standardiseret opsætning og kvantificering protokol, hvilket har ført til problemer med reproducerbarhed 7,10. Mange af de ændringer og optimeringer for at forbedre scratch-analysen har reduceret disse ...

Cellemigration meget koordineret og afgørende for mange fysiologiske processer, såsom vævsudvikling, reparation og regenerering, samt patologiske processer, såsom cancer metastase og arteriosklerose 1. En forståelse af celle migration er særligt relevant for nye behandlingsformer, der anvendes til at reparere beskadigede væv og behandle patologiske tilstande, herunder celle transplantation teknologi og kunstig væv podning 2. I betragtning af den nuværende interesse i rollen som mesenkymale stromaceller (MSC&#39;er) til at mediere vævsreparation 3, kvantificering af vandrende kapacitet i disse celler ved anvendelse af et assay, der er strengt kvantificerbar, tilpasningsdygtige og omkostningseffektiv er af interesse. Det er vigtigt, skal et sådant assay være tilstrækkelig følsom til at påvise relativt små ændringer i cellulære vandrende kapacitet efter skader. Nuværende metoder til kvantificering celle migration, herunder scratch assays, trans-brønds migration assays (Boyden chAmbers), micropillar arrays og celle udelukkelse zone analyser i besiddelse af en række begrænsninger i reproducerbarhed, customizability, kvantificering, og omkostningseffektivitet 1,4,5. Den optimerede scratch-analysen er beskrevet her demonstrerer robuste resultater, kvantificerbare og image-baserede analyse kapaciteter, omkostningseffektivitet og tilpasningsevne til andre applikationer. Scratch assays er blevet anvendt i flere kapacitet til at vurdere cellemigration og proliferation under forskellige forsøgsbetingelser 5. Assayet indebærer podning de udpegede celler, dyrke dem til fuldstændig eller næsten konfluens, og skrabe den resulterende monolag med en steril nål eller pipettespids 6. Analyse er mest almindeligt udføres ved at sammenligne bredden af bunden over flere tidspunkter på tilfældigt udvalgte steder 7-9. På trods af sin fremtrædende brug, er bunden analysen forvirrede af problemer med reproducerbarhed og kvantificering. Variabilitet igeneration af bunden ikke kun ændrer mikromiljø af cellerne, men kan også hæmme cellemigration ved at beskadige pladens overflade og den underliggende ekstracellulære matrix 5. Analyser ofte gennemført over 7 til 12 timer, men for cellelinjer viser langsommere migration og længere assay gange, spredning bliver en confounding variabel 7,10. Endelig kan senescentceller genereret af skrabe proces frigive faktorer, der forstyrrer den ekstracellulære signalering kræves for at lukke hullet i monolag 1. Optimering af bunden analysen kræver oprettelsen af ​​en sammenhængende hul, ikke interfererer med overfladeegenskaber, minimere assay tidslængde, og forhindre uønsket celledød under manipulation. Proppen baseret assay er en optimering af en celle udelukkelse zone assay. Dette assay anvender en prop placeret i midten af ​​borehullet, der udelukker cellevækst, men tillader celler at være belagt rundt om den centrale udelukkelse zone.For at vurdere migration, proppen fjernes, og den resulterende udelukkelse zone tilvejebringer en overflade for migration at forekomme. Det er imidlertid vanskeligt at tilpasse eller tilpasse 10 dette assay, og for nogle anvendelser kan denne teknik også omkostningskrævende. I modsætning til scratch assays og deres derivater, trans-brønd migration analyser (eller Boyden kammer analyser) vurdere migrationen ved at kvantificere antallet af celler, der bevæger sig fra det ene kammer, gennem en mikroporøs filter membran, ind i et kammer, der indeholder kemotaktiske agenter 8,11, 12. Denne teknik har begrænset anvendelighed til adhærente celler som MSC&#39;er fordi det efter migration gennem den porøse membran, adhærerer til membranen side udsat for de kemotaktiske midler og kan være svært præcist at kvantificere. Mens assayet er i stand til at undersøge nogle tredimensionale migrationsmønstre, de begrænsede celletyper, for hvilke det er i stand til præcist at kvantificere cellemigration begrænser dets anvendelighed 10. Et andet alternativ til bunden assays anvender et mikro-søjle array, som måler cellulær motilitet gennem et tredimensionalt rum ved hjælp af cellernes evne til at deformeres og migrere ind i array som et surrogat. Polydimethlysiloxane (PDMS) elastomerer hærdet i en præcis form og behandlet med ozon og fibronectin frembringer en homogen og ikke-nedbrydelige mikromiljø. Afstand mikro-søjle kan også varieres efter behov for at måle cellernes evne til at komme ind i arrayet 4. Formen er skabt gennem dyb reaktiv ion ætsning af siliciumskiver til at skabe en negativ version af high-aspekt forholdet gruppe 13. Mens analysen styrkes af dets customizability, evne til at modellere tredimensionelle migration og analyse gennem direkte visualisering af migrerende celler, svært ved at skabe mikro-søjle arrays økonomisk hindrer dens udbredte anvendelse. Den optimerede scratch-analysen er beskrevet i denne protokol giver en effektiv, cost-effektive fremgangsmåde til fremstilling af konsistente ridser, der kan analyseres ved frit tilgængeligt software. I stedet for simple bredde målinger foretaget på tværs af bunden før og efter celle migration, software gør det muligt for brugeren at bestemme det samlede scratch områder før og efter migrering. Denne avancement begrænser spørgsmålet om at forsøge at fastslå, hvor bunden af ​​breddemålinger skal tages, og om bredden af ​​bunden er ensartet langs dens længde. Desuden er omhyggelig optimering af celleantal, cellekonfluens og typen og graden af ​​skade påført på cellerne diskuteret med henblik på yderligere at optimere analysen.

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			<td height="22" style="height:22px;width:527px;">DMEM (high glucose, no added glutamine)</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">31053-036</td>
			<td style="width:307px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fetal Bovine Serum, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3091002</td>
			<td style="width:307px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Antibiotic/antimycotic, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SV3007901</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:527px;">Glutamine, 200mM, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3003401</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">TypeLE cell dissociation reagent</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">12563-01</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Dulbecco&#39;s Phosphate Buffered Saline, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3002802</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Falcon standard 60mm tissue culture dishes</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">08-772B</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fisherbrand Sterile 200ul pipet tips</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td>02-707-502</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Inverted light microscope with camera attachment</td>
			<td style="width:221px;">Variable</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
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		<tr height="38">
			<td height="38" style="height:39px;width:527px;">ImageJ software&#160;</td>
			<td style="width:221px;">http://imagej.nih.gov/ij/</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
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		<tr height="85">
			<td height="85" style="height:85px;width:527px;">MRI_Wound_Healting plugin</td>
			<td style="width:221px;"><a href="http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool">http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool</a></td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Statistical analysis software</td>
			<td style="width:221px;">Microsoft Excel</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
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optimization of the wound scratch assay to detect changes in murine mesenchymal stromal cell migration after damage by soluble cigarette smoke extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

De scratch analyser præsenteres her blev udført ved hjælp af murine lunge hjemmehørende mesenkymale stromale celler (LR-MSC&#39;er), og isoleret som refereret i protokollen noter. LR-MSC&#39;er blev podet ved en densitet på 500.000 celler i en 60 mm vævskulturskål og dyrket til 90% konfluens i 48 timer. For at generere skader, blev 4% CSE (beskrevet ovenfor) inkuberes med cellerne i 24 timer efter den første podning udløb, men inden bunden assay. For hvert assay blev bunden fremkommet ved en 200 pi pipettespids...

Watch this Scientific Journal Video about Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract at JoVE

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Optimering af såret Scratch-analysen til at påvise ændringer i murine Mesenchymale stromacelle Migration efter skade ved Opløselig Cigaret røgekstrakt

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

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Aorta Ring Co kultur Assay: Et praktisk værktøj til at vurdere angiogene af mesenchymale stromale celler In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Celle sammenlægning Assays til at evaluere bindingen af Drosophila Notch med Trans-ligander og dens hæmning af Cis-ligander

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Visualisering af Tangential celle Migration i den udviklende Chick optiske Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...