Name: protrusion force microscopy: a method to quantify forces developed by cell protrusions
Uploaded: 2018-06-16T14:00:00
Description: Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

Forfatterne er takknemlig til Anna Labernadie, Guillaume Charrière og Patrick Delobelle for deres første bidrag til dette arbeidet og Matthieu Sanchez og Françoise Viala for deres hjelp med filming og redigering. Dette arbeidet har blitt støttet av l'Agence Nationale de la Recherche (ANR14-CE11-0020-02), la Fondation pour la Recherche Médicale (FRM DEQ2016 0334894), INSERM Plan kreft, Fondation Toulouse kreft og menneskelige Frontier Science Program (RGP0035/2016).

1. forberedelse av Formvar-belagt rutenett
<ol><li>Rengjør elektronmikroskop rutenett med ren aceton og tørk dem på filter papir. Rengjør et mikroskop lysbilde med ren etanol, tørk med linsen papir og fjerne støv med en vifte.</li>
	<li>Plass en etanol-renset objektglass loddrett i trakten av filmen casting enhet som inneholder en løsning av Formvar i den nedre delen. Dekk toppen av trakten.</li>
	<li>Pumpe 100 mL Formvar løsning (0,5% i etylen dichloride) med atomizer pære inntil nivået når to tredjedeler a...

<ol>
	<li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Cells+Feel+and+Respond+to+the+Stiffness+of+Their+Substrate.">Tissue Cells Feel and Respond to the Stiffness of Their Substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Paszek, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+Homeostasis+and+the+Malignant+Phenotype.">Tensional Homeostasis and the Malignant Phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Elasticity+Directs+Stem+Cell+Lineage+Specification.">Matrix Elasticity Directs Stem Cell Lineage Specification.</a> Cell. 126, 677-689 (2006).</li><li>Gilbert, P. M., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Adaptation+to+Biomechanical+Stress+across+Length+Scales+in+Tissue+Homeostasis+and+Disease.">Cellular Adaptation to Biomechanical Stress across Length Scales in Tissue Homeostasis and Disease.</a> Seminars in Cell &amp; Developmental Biology. 67, 141-152 (2017).</li><li>Vining, K. H., Mooney, D. 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Force Generation and Mechanosensing Activity of Human Macrophage Podosomes.</a> Nature Communications. 5, 5343 (2014).</li><li>Proag, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+Together:+Spatial+Synchrony+in+the+Force+and+Actin+Dynamics+of+Podosome+First+Neighbors.">Working Together: Spatial Synchrony in the Force and Actin Dynamics of Podosome First Neighbors.</a> ACS Nano. 9, 3800-3813 (2015).</li><li>Proag, A., Bouissou, A., Vieu, C., Maridonneau-Parini, I., Poincloux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Force+and+Spatial+Dynamics+of+Macrophage+Podosomes+by+Multi-Particle+Tracking.">Evaluation of the Force and Spatial Dynamics of Macrophage Podosomes by Multi-Particle Tracking.</a> Methods. 94, 75-84 (2016).</li><li>Cougoule, C., et al. <a 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Materialegenskaper
Valg av materiale for deformerbare membranen, i vårt tilfelle Formvar, må oppfylle noen få krav. Materialet må være gjennomsiktig til synlig lys og utstillingen begrenset auto fluorescens å tillate observasjoner i lyse og fluorescens mikroskopi. Råhet av tynne filmen må være godt under 10 nm å unngå noen topografiske effekt på celleadhesjon og å tillate klart observasjon av de celle-indusert utstikkende deler av AFM tenkelig. Til slutt, stivhet ...

Dyreceller samhandle fysisk med matrisen og de andre cellene som utgjør deres miljø1. Dette er nødvendig for dem å migrere, internalisere organer, hente eksterne informasjon eller skille. I slike prosesser, cellen må generere mekaniske krefter og, som mange studier har vist de siste årene, muligheten for en celle for å generere styrker og sonde omgivelsene påvirker virkemåten biologiske, regi for eksempel spredning eller differensiering2,3. Igjen, er måling av mobilnettet styrker en stor hjelp til studere regulering av styrkegenerering og forstå dens innblanding i cellen atferd og vev skjebne4,5.
De siste årene har vært vitne til utviklingen av mange teknikker å måle kreftene som en celle kan øve på sin omgivelsene6. Fleste av disse har vært medvirkende i avslørende trekkraft styrker at celler utøve de dra på mobile sonder eller en deformerbare substrat. Men de mekaniske kreftene involvert i protrusion ekstracellulære Environment lider av mangel på måling teknikker og er ennå ikke godt preget.
For å overkomme denne begrensningen, presenterer vi en metode for å måle styrker utøvde ortogonalt til underlaget. Det består i plating levende celler på et tynt elastisk ark som kan deformere ortogonale retning og gjør det mulig å måle substrat deformasjon av cellene og utlede kreftene som er involvert. Substrat topografi måles med nanoskala oppløsning bruker atomic force mikroskopi og evaluering av styrker fra deformasjon avhengig av kunnskap om geometrien av protrusive cellulære strukturer7,8, 9.
Her beskriver vi oppsettet og dens anvendelse å måle styrkene generert av podosomes, protrusive vedheft strukturer av makrofager for deres mesenchymal overføring i tredimensjonale omgivelser10,11, 12,,13,,14,,15,,16,,17. Vi tror at denne teknikken vil fremme forståelsen av styrkegenerering og dens regulering i mange mobilnettet prosesser som involverer protrusion.

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		<tr height="51">
			<td height="51" style="height:51px;width:216px;">200 mesh nickel grids</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">G200-Ni</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">Filter paper</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1001-055</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">Microscope slides</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">10235612</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">White stickers 26 x 70 mm</td>
			<td style="width:71px;">Avery</td>
			<td style="width:119px;">DP033-100</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Film casting device with valve in its outlet</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">71305-01</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Razorblades</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">72000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">1.08543.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetone</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">20066.321</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Formvar 0.5% solution in ethylene dichloride</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">12 mm coverslips</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-0666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">Axiovert 200</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Atomic Force Microscope</td>
			<td style="width:71px;">JPK Instruments</td>
			<td style="width:119px;">NanoWizard III</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Temperature-controlled sample holder&#160;</td>
			<td style="width:71px;">JPK Instruments</td>
			<td style="width:119px;">BioCell</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Silicon nitride cantilever with a nominal spring constant of 0.01 N/m</td>
			<td style="width:71px;">Veeco Instruments</td>
			<td style="width:119px;">MLCT-AUHW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double-sided adhesive tape</td>
			<td style="width:71px;">APLI AGIPA</td>
			<td style="width:119px;">118100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31870-025</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">FCS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7524</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">HEPES&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm glass-bottom Petri dishes</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">FD35-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

protrusion force microscopy: a method to quantify forces developed by cell protrusions

I mange biologiske sammenhenger må dyreceller bruke fysisk miljøet ved å utvikle mekaniske krefter. Blant disse, trekkraft styrker har vært godt karakterisert, men det er en mangel på teknikker slik at måling av protrusion styrkene utøves av cellene ortogonalt deres substrat. Vi utviklet en eksperimentelle oppsett å måle protrusion styrkene utøves av tilhenger cellene på deres substrat. Celler belagt på en kompatibel Formvar deformere dette underlaget og resulterende topografi er tilordnet av atomic force mikroskopi (AFM) i nanometer skala. Force verdier er deretter trukket ut fra en analyse av deformasjon profil basert på geometrien av protrusive cellular strukturer. Derfor kan styrker utøves av individet utstående enheter av en levende celle måles over tid. Denne teknikken gjør studiet av styrkegenerering og dens regulering i mange mobilnettet prosesser som involverer protrusion. Her beskriver vi programmet å måle protrusive krefter generert av podosomes dannet av menneskelig makrofager.

Over protokollen beskriver hvordan å forberede det eksperimentelle setup å kvantifisere protrusion styrker av macrophage podosomes på et Formvar substrat. Dette oppnås ved hjelp av AFM er illustrert i figur 1.
Når du analyserer en topografiske bilde av buler under podosomes ved hjelp av JPK databehandling programvaren, skal et tredje grad polynom passer uavhengig trekkes fra hver skanning linje...

Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

Her, detalj vi eksperimentelle teknikker brukes til å evaluere protrusion styrkene som podosomes gjelder på en kompatibel film, fra forberedelse av filmen til automatisk analyse av topografiske bilder.

Protrusion Force mikroskopi: En metode for å kvantifisere styrker utviklet av cellen utstikkende deler

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

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Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

Live-celle bildebehandling av encellede matriser (LISCA) - en allsidig teknikk for å kvantifisere cellulær kinetikk

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

Måling av cellekantprotruderingsdynamikk under spredning ved hjelp av live-cellemikroskopi

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...