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 Anna Labernadie, Guillaume Charrière og Patrick Delobelle taknemmelig for deres indledende bidrag til dette arbejde og Matthieu Sanchez og Françoise Viala for deres hjælp med video filme og redigere. Dette arbejde er blevet støttet af l'Agence Nationale de la Recherche (ANR14-CE11-0020-02), la Fondation pour la Recherche Médicale (FRM DEQ2016 0334894), INSERM planlægger kræft, Fondation Toulouse kræft og Human Frontier Science Program (RGP0035/2016).

1. forberedelse af Formvar-belagt gitre
<ol><li>Ren elektronmikroskopi gitre med ren acetone og tør dem på filtrerpapir. Derefter rengøre et objektglas med ren ethanol, tørres af med en linse papir og fjerne støv med en blæser.</li>
	<li>Sted en ethanol-rengjort glas dias lodret i tragten af en film casting enhed indeholder en opløsning af Formvar i den nederste del. Dække toppen af tragten.</li>
	<li>Pumpe 100 mL Formvar opløsning (0,5% i ethylen dichloride) med forstøver pære, indtil niveauet når to tredj...

<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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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Architecture+Dictates+Three-Dimensional+Migration+Modes+of+Human+Macrophages:+Differential+Involvement+of+Proteases+and+Podosome-Like+Structures.">Matrix Architecture Dictates Three-Dimensional Migration Modes of Human Macrophages: Differential Involvement of Proteases and Podosome-Like Structures.</a> Journal of Immunology. 184, 1049-1061 (2010).</li><li>Verollet, C., 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=Hiv-1+Reprograms+the+Migration+of+Macrophages.">Hiv-1 Reprograms the Migration of Macrophages.</a> Blood. 125, 1611-1622 (2015).</li><li>Bouissou, A., et al. <a target="_blank" 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Materialeegenskaber
Valget af materiale til den deformerbare membran, i vores tilfælde Formvar, skal opfylde nogle krav. Materialet skal være transparent for synligt lys og udviser begrænset auto fluorescens for at tillade observationer i lysfelt og Fluorescens mikroskopi. Ruhed af den tynde film skal være et stykke under 10 nm til at undgå enhver topografiske effekt på celle vedhæftning og tillade klare observation af de celle-induceret fremspring af AFM billeddannelse...

Animalske celler interagere fysisk med matrix og de andre celler, der udgør deres miljø1. Dette er nødvendigt for dem at migrere, internalisere organer, erhverve eksterne oplysninger, eller differentiere. I sådanne processer, cellen skal generere mekaniske kræfter og som talrige undersøgelser har vist i de seneste år, evne til en celle for at generere styrker og sonde omgivelserne påvirker dens biologiske opførsel, lede f.eks spredning eller differentiering2,3. Igen, er måling af cellulære styrker en stor støtte til at undersøge regulering af styrkegenerering og forstår dets konsekvenser i celle adfærd og væv skæbne4,5.
De seneste år har været vidne til udviklingen af mange teknikker til at måle de kræfter, som en celle kan øve på dens miljø6. Fleste af disse har været medvirkende til at afsløre trækkraften styrker at celler udøve som de trækker på mobile sonder eller en deformerbar substrat. Men de mekaniske kræfter involveret i fremspring i det ekstracellulære miljø lider af en mangel på standardiserede måleteknikker og er til dato ikke godt karakteriseret.
For at overvinde denne begrænsning, præsenterer vi en metode til at måle kræfter udøver ortogonalt til underlaget. Det består i plating levende celler på en tynd elastisk ark, som kan deformere i ortogonale retning, hvilket gør det muligt at måle substrat deformation af celler og udlede kræfter involveret. Substrat topografi er målt med nanoskala opløsning ved hjælp af atomic force mikroskopi og evalueringen af styrker fra deformation bygger på viden om geometri af protrusive cellestrukturer7,8, 9.
Her, beskrive vi setup og dens anvendelse til at måle de kræfter, der genereres af podosomes, protrusive vedhæftning strukturer dannet af makrofager for deres mesenchymale migration i tre-dimensionelle miljøer10,11, 12,13,14,15,16,17. Vi mener, at denne teknik vil fremme forståelsen af styrkegenerering og dens regulering i de mange cellulære processer der involverer fremspring.

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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>
		<tr height="21">
			<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 sammenhænge skal animalske celler interagere fysisk med deres miljø ved at udvikle mekaniske kræfter. Blandt disse, trækkraft styrker har været godt karakteriseret, men mangler teknikker tillader måling af fremspring styrker udøves af celler ortogonalt til deres substrat. Vi designede en eksperimentel setup at måle fremspring styrker udøves af vedhængende celler på deres substrat. Celler belagt på en kompatibel Formvar ark deformere dette substrat og den resulterende topografi er tilknyttet ved atomic force mikroskopi (AFM) på nanometer skala. Gældende værdier er derefter udvundet fra en analyse af den deformation profil baseret på geometrien af protrusive cellulære strukturer. Derfor kan de kræfter, der udøves af de enkelte fremspringende enheder af en levende celle måles over tid. Denne teknik gør det muligt studiet af styrkegenerering og dens regulering i de mange cellulære processer der involverer fremspring. Her beskriver vi dets anvendelse til at måle de protrusive styrker genereret af podosomes dannet af menneskelige makrofager.

Den ovennævnte protokol beskriver hvordan du forbereder det eksperimentelle setup til at kvantificere fremspring kræfter af makrofag podosomes på en Formvar substrat. Dette er opnået ved hjælp af AFM og er illustreret i figur 1.
Når du analyserer en topografiske billede af buler under podosomes ved hjælp af KURIA databehandling software, skal et tredje grad polynomium passer trækkes fra hver...

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, detalje vi de eksperimentelle teknikker, der anvendes til at evaluere de fremspring kræfter, der podosomes anvendelse på en kompatibel film, fra forberedelse af filmen til den automatiserede analyse af topografiske billeder.

Fremspring Force mikroskopi: En metode til at kvantificere kræfter udviklet af celle fremspring

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

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

Live-cell Imaging af single-cell arrays (LISCA) - en alsidig teknik til at kvantificere Cellular Kinetics

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 af cellekantfremspringsdynamik under spredning ved hjælp af levende cellemikroskopi

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