Name: quantitative analysis of cell edge dynamics during cell spreading
Uploaded: 2021-05-22T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Dette arbejde blev støttet af Connaught Fund New Investigator Award til S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (tilskud RGPIN-2015-05114 og RGPIN-2020-05881), University of Manchester og University of Toronto Joint Research Fund og University of Toronto XSeed Program.

1. Cellesåning
BEMÆRK: Den beskrevne cellespredningsprotokol blev udført ved hjælp af musefosfosiske fibroblaster (MEF'er), der udtrykker PH-Akt-GFP (en fluorescerende markør for PIP3/PI(3,4)P2). Denne cellelinje blev genereret ved genomisk at integrere en ekspressionskonstruktion til PH-Akt-GFP (Addgene #21218) ved CRISPR-medieret genredigering. Andre lysstofrør, der udtrykkes forbigående eller integreres i genomet, kan dog også bruges i denne analyse. For optimal ...

<ol>
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L., 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=Cell+migration+without+a+lamellipodium.">Cell migration without a lamellipodium.</a> The Journal of Cell Biology. 168 (4), 619-631 (2005).</li><li>Dimchev, V., 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=Induced+Arp2/3+Complex+Depletion+Increases+FMNL2/3+Formin+Expression+and+Filopodia+Formation.">Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.</a> Frontiers in Cell and Developmental Biology. 9, 634708 (2021).</li><li>Leithner, 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=Diversified+actin+protrusions+promote+environmental+exploration+but+are+dispensable+for+locomotion+of+leukocytes.">Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.</a> Nature cell biology. 18 (11), 1253-1259 (2016).</li><li>Giannone, G., Dubin-Thaler, B. J., D&#246;bereiner, H. -. G., Kieffer, N., Bresnick, A. R., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+Lamellipodial+Contractions+Correlate+with+Rearward+Actin+Waves.">Periodic Lamellipodial Contractions Correlate with Rearward Actin Waves.</a> Cell. 116 (3), 431-443 (2004).</li><li>Dubin-Thaler, B. 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=Quantification+of+Cell+Edge+Velocities+and+Traction+Forces+Reveals+Distinct+Motility+Modules+during+Cell+Spreading.">Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading.</a> PLoS ONE. 3 (11), 3735 (2008).</li><li>Suraneni, P., Rubinstein, B., Unruh, J. R., Durnin, M., Hanein, D., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arp2/3+complex+is+required+for+lamellipodia+extension+and+directional+fibroblast+cell+migration.">The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration.</a> The Journal of cell biology. 197 (2), 239-251 (2012).</li><li>Wang, 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=Deconvolution+of+subcellular+protrusion+heterogeneity+and+the+underlying+actin+regulator+dynamics+from+live+cell+imaging.">Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging.</a> Nature Communications. 9 (1), 1688 (2018).</li><li>Dimchev, G., 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=Lamellipodin+tunes+cell+migration+by+stabilizing+protrusions+and+promoting+adhesion+formation.">Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation.</a> Journal of cell science. 133 (7), 239020 (2020).</li><li>Burnette, D. T., 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=A+role+for+actin+arcs+in+the+leading-edge+advance+of+migrating+cells.">A role for actin arcs in the leading-edge advance of migrating cells.</a> Nature cell biology. 13 (4), 371-381 (2011).</li><li>Yamada, K. M., Kennedy, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dualistic+nature+of+adhesive+protein+function:+fibronectin+and+its+biologically+active+peptide+fragments+can+autoinhibit+fibronectin+function.">Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function.</a> The Journal of Cell Biology. 99 (1), 29-36 (1984).</li><li>Cai, Y., 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=Nonmuscle+Myosin+IIA-Dependent+Force+Inhibits+Cell+Spreading+and+Drives+F-Actin+Flow.">Nonmuscle Myosin IIA-Dependent Force Inhibits Cell Spreading and Drives F-Actin Flow.</a> Biophysical Journal. 91 (10), 3907-3920 (2006).</li><li>Humphries, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+assays.">Cell adhesion assays.</a> Molecular Biotechnology. 18 (1), 57-61 (2001).</li><li>Cavalcanti-Adam, E. A., Volberg, T., Micoulet, A., Kessler, H., Geiger, B., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Spreading+and+Focal+Adhesion+Dynamics+Are+Regulated+by+Spacing+of+Integrin+Ligands.">Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands.</a> Biophysical Journal. 92 (8), 2964-2974 (2007).</li><li>Dubin-Thaler, B. J., Giannone, G., D&#246;bereiner, H. -. G., Sheetz, M. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Migration,+Developmental+Methods+and+Protocols.">Cell Migration, Developmental Methods and Protocols.</a> Methods in molecular biology. 294, 55-68 (2004).</li><li>Raucher, D., 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=Phosphatidylinositol+4,5-Bisphosphate+Functions+as+a+Second+Messenger+that+Regulates+Cytoskeleton-Plasma+Membrane+Adhesion.">Phosphatidylinositol 4,5-Bisphosphate Functions as a Second Messenger that Regulates Cytoskeleton-Plasma Membrane Adhesion.</a> Cell. 100 (2), 221-228 (2000).</li><li>Machacek, M., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphodynamic+Profiling+of+Protrusion+Phenotypes.">Morphodynamic Profiling of Protrusion Phenotypes.</a> Biophysical Journal. 90 (4), 1439-1452 (2006).</li><li>Zack, G. 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Den beskrevne cellespredningsanalyse giver mulighed for kontinuerlig sporing af morfologiske ændringer(f.eks. cellestørrelse og -form) og cellekantbevægelser(dvs. fremspringshastighed og tilbagetrækningsfrekvens), som er funktioner, der mangler i de fleste cellespredningsprotokoller19,24. Mens almindeligt anvendte endepunkt celle sprede assays giver mulighed for bestemmelse af cellespredning hastighed, disse assays undlader at løse den tids...

Lamellipodial fremspring er fremtrædende cytoskeletale strukturer dannet på forsiden af en migrerende celle. I lamellipodia skaber polymerisering af actin ved hjælp af Arp2/3-komplekset og forminer et hurtigt voksende forgrenet actin meshwork, der skubber mod plasmamembranen1,2. Den skubbekraft , der genereres af det actin meshwork , driver fysisk cellen fremad1,3,4,5. Udtømning af Arp2/3-komplekset eller afbrydelse af signalveje , der er afgørende for lamellipodiale fremspring , forringer oftecellemigrationen 6, 7. Selv om migration af lamellipodia-mangelfulde celler også er blevet rapporteret8,9, betydningen af lamellipodi i celle migration er tydeligt som udtømning af denne fremspringende struktur gennemsyrer cellens evne til at bevæge sig gennem komplekse biologiske mikromiljøer6,10.
En væsentlig hindring for at forstå reguleringen af lamellipodi i migrerende celler er den naturlige variation i lamellipodial protrusion kinetik, størrelse og form11,12,13,14. Desuden har nylige undersøgelser vist, at lamellipodia udviser kompleks fremspringende adfærd, herunder svingende, periodiske og accelererende fremspring14,15. Sammenlignet med de meget variable lamellipodier af migrerende celler6,16, lamellipodia dannet under cellespredning er mereensartet 12. Da fremspringende aktivitet spredning og migrerende celler er drevet af identiske makromolekylære forsamlinger, som omfatter en forgrenet actin netværk, kontraktil actomyosin bundter, og integrin-baserede celle-matrix vedhæftninger17,18, sprede celler er blevet udbredt som en model for at undersøge reguleringen af lamellipodia dynamik.
Cellespredning er en dynamisk mekanokemisk proces , hvor en celle i suspension først klæber til et substrat gennem integrinbaserede vedhæftninger17,19,20 og derefter spredes ved at udvide actin-baserede fremspring21,22,23. Under spredningsfasen stikker lamellipodier, der stammer fra cellekroppen, isotropisk og vedvarende med lidt eller ingen tilbagetrækning eller hingst12. De mest anvendte cellespredningsprotokoller er endpoints assays, hvor spredning af celler rettes på forskellige tidspunkter efter plating19,24. Disse assays, selv om hurtig og enkel, er begrænset i deres diagnostiske magt til at opdage ændringer i de dynamiske funktioner i lamellipodia. For at bestemme de molekylære mekanismer, der styrer lamellipodiadynamik, var Sheetz-gruppen banebrydende for brugen af kvantitativ analyse af levende spredeceller og afdækkede mange grundlæggende egenskaber ved cellekantfremspring11,12,22. Disse undersøgelser har vist, at levende celle sprede analyse er en robust og kraftfuld teknik i værktøjskassen i en celle biologi laboratorium. På trods af dette er en detaljeret protokol og open source-beregningsværktøj til en spredning af levende celler i øjeblikket ikke tilgængeligt for cellebiologisamfundet. Til dette formål skitserer vores protokol procedurerne for billeddannelse af levende sprede celler og giver et automatiseret billedanalyseværktøj. For at validere denne metode brugte vi Arp2/3-hæmning som en eksperimentel behandling og viste, at hæmning af Arp2/3-kompleksets funktion ikke arresterede cellespredning, men forårsagede en betydelig reduktion i cellefremspringshastigheden samt stabiliteten af cellekantfremspring, hvilket gav anledning til takkede cellekanter. Disse data viser, at kombinationen af live-celle billeddannelse og automatiseret billedanalyse er et nyttigt værktøj til at analysere cellekant dynamik og identificere molekylære komponenter, der regulerer lamellipodia.

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			<td>0.05% Trypsin (0.05%), 0.53 mM EDTA</td>
			<td>Wisent Bioproducts</td>
			<td>325-042-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>10.0 cm Petri Dish, Polystyrene, TC Treated, Vented</td>
			<td>Starstedt</td>
			<td>83.3902</td>
			<td></td>
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		<tr>
			<td>15 mL High Clarity PP Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
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			<td>1-Well Chamlide CMS for 22 mm x 22 mm Coverslip</td>
			<td>Quorum Technologies</td>
			<td>CM-S22-1</td>
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			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
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			<td>50 mL Centrifuge Tube, Transparent, Plug Seal</td>
			<td>Nest</td>
			<td>602002</td>
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			<td>6.0 cm Cell Culture Dishes Treated for Increased Cell Attachment, Sterile</td>
			<td>VWR</td>
			<td>10861-658</td>
			<td></td>
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			<td>Arp2/3 Complex Inhibitor I, CK-666</td>
			<td>Millipore Sigma</td>
			<td>182515</td>
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			<td>Camera, Prime 95B-25MM</td>
			<td>Photometrics</td>
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			<td>Dimethyl Sulfoxide, Sterile</td>
			<td>BioShop</td>
			<td>DMS666</td>
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			<td>DMEM, 1x, 4.5 g/L Glucose, with L-Glutamine, Sodium Pyruvate and Phenol Red</td>
			<td>Wisent Bioproducts</td>
			<td>319-005 CL</td>
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			<td>DMEM/F-12, HEPES, No Phenol Red</td>
			<td>Gibco</td>
			<td>11039021</td>
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			<td>D-PBS, 1X</td>
			<td>Wisent Bioproducts</td>
			<td>311-425 CL</td>
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			<td>Fetal Bovine Serum</td>
			<td>Wisent Bioproducts</td>
			<td>080-110</td>
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			<td>Fiji Software</td>
			<td>ImageJ</td>
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			<td></td>
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			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
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			<td>Human Plasma Fibronectin Purified Protein 1 mg</td>
			<td>Millipore Sigma</td>
			<td>FC010</td>
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			<td>Cargille</td>
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			<td>Wisent Bioproducts</td>
			<td>609-065-EL</td>
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			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
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			<td>Micro Cover Glasses, Square, No. 11/2 22 x 22 mm</td>
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			<td>Microscope Body, Eclipse Ti2-E</td>
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			<td>Objective, CFI Plan Apo Lambda 60X Oil</td>
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			<td>Spinning Disk, Crest Light V2</td>
			<td>CrestOptics</td>
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			<td>Spyder</td>
			<td>Anaconda</td>
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			<td>Stage top incubator</td>
			<td>Tokai Hit</td>
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			<td>Statistics Software, Prism</td>
			<td>GraphPad</td>
			<td></td>
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			<td>Tweezers, Style 2</td>
			<td>Electron Microscopy Sciences</td>
			<td>78326-42</td>
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quantitative analysis of cell edge dynamics during cell spreading

Cellespredning er en dynamisk proces, hvor en celle, der er ophængt i medier, fastgøres til et substrat og samkopierer sig selv fra en afrundet til en tynd og spredt form. Efter celle-substrat vedhæftet fil, cellen danner et tyndt ark lamellipodia stammer fra cellekroppen. I lamellipodien polymeriseres kugleformede actin (G-actin) monomerer til et tæt filamentous actin (F-actin) meshwork, der skubber mod plasmamembranen, hvilket giver de mekaniske kræfter, der kræves for, at cellen kan sprede sig. Især de molekylære spillere, der styrer actin polymerisering i lamellipodia er afgørende for mange andre cellulære processer, såsom celle migration og endokytose. 
Da sprede celler danner kontinuerlig lamellipodi, der spænder over hele cellen periferien og vedvarende udvide udad, celle sprede assays er blevet et effektivt redskab til at vurdere kinetik lamellipodial fremspring. Selv om flere tekniske implementeringer af cellespredningsanalysen er blevet udviklet, mangler der i øjeblikket en detaljeret beskrivelse af arbejdsgangen, som vil omfatte både en trinvis protokol og beregningsværktøjer til dataanalyse. Her beskriver vi de eksperimentelle procedurer i cellespredningsanalysen og præsenterer et open source-værktøj til kvantitativ og upartisk analyse af cellekantdynamik under spredning. Når denne protokol kombineres med farmakologiske manipulationer og/eller genhæmningsteknikker, kan den tilpasses en storstilet skærm af molekylære afspillere, der regulerer lamellipodiale fremspring.

Ovenstående protokol beskriver de eksperimentelle procedurer for levende celle billeddannelse af sprede celler og et beregningsværktøj til kvantitativ analyse af cellespredning dynamik. Beregningsværktøjet kan bruges i et lav- eller højoverførselsformat til at identificere de molekylære spillere, der regulerer actin-polymeriseringsmaskineriet ved cellekanten.
Den skematiske repræsentation af forsøgsprocedurerne er vist i figur 1. Cellen sprede assay blev...

Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

I denne protokol præsenterer vi de eksperimentelle procedurer i en cellespredningsanalyse, der er baseret på levende cellemikroskopi. Vi leverer et open source beregningsværktøj til uvildig segmentering af fluorescerende mærkede celler og kvantitativ analyse af lamellipodia dynamik under cellespredning.

Kvantitativ analyse af cellekantdynamik under cellespredning

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Protokollen, der er beskrevet i denne video, definerer de eksperimentelle procedurer, der kræves til billeddannelse i naturlig størrelse af spredning af celler og brugen af et beregningsværktøj, der kvantificerer celler, der spreder dynamikken på en upartisk og automatiseret måde. Cellespredningsaktivet giver mulighed for kontinuerlig sporing af cellekantbevægelse og morfologiske ændringer under cellespredning, hvilket er en funktion, der mangler i de fleste eksisterende cellespredningsanalyser. Denne protokol implementerer også brugen af automatiseret computerbehandling og -analyse, der giver oplysninger om cellecirkulæritet, område- og fremspringsudtrækscyklusser for spredning af celler.En sådan automatiseret behandling reducerer bias i dataanalyse og giver en robust metode til at analysere cellespredningsdynamik for et stort antal celler. Når den kombineres med medicinske behandlinger eller genvidenskab og -teknikker, er denne protokol modtagelig for store hastigheder af molekylære spillere, der regulerer cellefremspring. For den givne protokol, De anvendte celler er mus embryonale fibroblaster, der genetisk indkode PH-Akt-GFP, som giver mulighed for fluorescerende tagging af plasmamembranen.Forud for begyndelsen af cellen sprede analyse, kultur en parabol af celler til 90% sammenløb. Når cellerne har opnået den rette sammenløb, flamme en 22 af 22 millimeter dække slip og læg den i en 35 millimeter cellekultur parabol. Dækslet skaledlerne belægges med fibronectin, der er fortyndet i PBS, til en koncentration på 2,5 mikrogram pr. milliliter.Placer skålen med dækslet glide ind i en 37 graders inkubator i en time. Efter en time indsugning af fibronectin og sørg for ikke at røre dækslet glide med pipette spidsen. Vask skålen med PBS ved forsigtigt at pipere rundt om dækslet to til tre gange.For celle såning, begynde med at aspirere cellekultur medier fra parabol af celler. Bagefter vaskes skålen med varm PBS. Tilsæt 650 mikroliter trypsin EDTA til skålen og vip skålen for at fordele enzymet jævnt.Placer skålen med trypsin i inkubatoren i et minut. Efter inkubation skal du først tilføje 10 milliliter cellekulturmedier i et centrifugerør. Derefter skal du hurtigt tilføje yderligere 10 milliliter medier i skålen for at slukke trypsin.For at fortynde de celler, der vil blive seedet på dækslet slip, pipette en milliliter af skålens indhold i centrifuge røret. Fra røret pipettes ca. 500 til 1.000 mikroliter af fortyndede celler i skålen med dækslet. Sørg for, at dækslet slip er på en 10% sammenløb eller 50.000 celler pr milliliter og justere mængden af fortyndede celler efter behov.Formålet med disse celler er at etablere et plan for fokus under billederhvervelse. Med de resterende celler i skålen, passage en femtedel af cellerne i en lille skål pr behandling. Disse vil være de celler, der vil blive analyseret for at sprede dynamik.Placer passage retter og dækslet slip parabol i en inkubator i otte til 24 timer. For at teste vigtigheden af Arp2/3 for cellespredning skal du først tilføje fem milliliter cellekulturmedier i en kontrol og et behandlingscentrifugerør. Derefter tilsættes 20 milliliter dmem, der mangler phenolrødt i hver af to større centrifugerør.Pipette enten stoffet CK-666, som er en hæmmer af Arp2/3 komplekset eller den kontrollerede behandling såsom DMSO i de små og store rør. For at begynde farmakologisk behandling af cellerne skal du fjerne de passageretter, der inkuberes natten over. Indsug cellekulturmedierne fra alle retterne, og vask opvasken med varm PBS.Tag de små rør, der indeholder enten CK-666 eller kontrol supplerede medier og tilsæt indholdet af det relevante rør i hver af passagen retter. Mærk hver af retterne med den korrekte lægemiddelbehandling, og placer derefter opvasken tilbage i inkubatoren i en ekstra time Efter en times inkubation supplerede stoffet medierne fra hver af retterne. Dernæst tilsættes varm PBS til alle retterne for grundigt at fjerne alle de resterende phenol røde medier.Tilsæt 230 mikroliter trypsin EDTA og læg opvasken i en inkubator i et minut. Fjern de trypsiniserede retter fra inkubatoren og fortsæt med at tilføje fem milliliter af lægemidlet suppleret dmem, der mangler phenol rød i et rør, der er udpeget som rør B.Derefter tilsættes yderligere fem milliliter af samme medie i den relevante skål for at slukke trypsin. Pipetter mediet op og ned flere gange for at fjerne alle cellerne fra skålen.Overfør alt indholdet af skålen til et andet rør, der allerede er udpeget som rør A.In for at forberede en cellefortynding, der er egnet til billeddannelse, overføre en milliliter celler fra rør A til rør B.Gentag alle fortyndingstrinnene for hver behandling. Placer alle rørene i inkubatoren i 45 minutter for at give cellerne mulighed for at komme sig efter trypsinisering. Sørg for, at alle dele af en celle magnetisk kammer, der kan rumme en 22 af 22 millimeter dække slip er blevet grundigt rengjort før brug.Fjern skålen med dækslet fra inkubatoren. Indsug cellekulturmediet, og vask dækslet med varm PBS. Fjern dækslet slip ved hjælp af et par pincet og forsigtigt lægge dækslet glide på bunden plade af det magnetiske kammer.Derefter afhente silikone pakning og læg den på toppen af dækslet slip. Fastgør hoveddelen af det magnetiske kammer på bundpladen. Der tilsættes en milliliter af dmem'en, der mangler phenolrød, svarende til den relevante behandling, til magnetkammeret.Tag et fnugfrit væv og dup forsigtigt kabinettet mellem hovedkroppen og bundpladen for at kontrollere eventuelle lækager. For at fuldføre det magnetiske kammer skal du sænke det gennemsigtige dæksel på hovedkroppen. Til sidst tørres bunden af dækslet med et væv sprøjtet med vand og en anden sprøjtes med 70% ethanol.Forvarm fase top inkubatoren af et konfokalt mikroskop til 37 grader. Placer det magnetiske kammer oven på målet. Før du erhverver spredningsdynamik, skal du sætte fokus på de allerede polariserede celler i den grønne fluorescerende kanal.Dette sikrer, at spredningscellerne vil være i fokus, når de fastgøres til dækslet. Fjern det gennemsigtige dæksel af det magnetiske kammer og pipetten 500 mikroliter fra rør B, der blev fjernet fra inkubatoren. Placer det gennemsigtige dæksel tilbage på toppen.At identificere celler, der er ideelle til cellespredningsanalyse, skal du søge efter grønne glorier, der repræsenterer celler, der endnu ikke er fastgjort til dækslet, eller som befinder sig i de tidligste stadier af den vedhæftede fil. Anskaf billeder, og gem filerne. Efter at have afsluttet billederhvervelsen af cellespredning, skal du begynde med at køre en kompatibel Python IDE som Spyder.Til beregningsanalyse af cellespredningsdynamik skal du finde og åbne den gui-fil til cellespredning, der findes i de relevante scriptpakker. Tryk på knappen Kør, som åbner panelet for analyse-GUI i baggrunden. Gui huser alle de indstillinger, der er nødvendige for analyse.Hvis du vil analysere celleområde og cirkularitet, skal du indtaste anskaffelsesfilen og de nødvendige indstillinger under fanen celleopslagsområde. Justeringer skal foretages afhængigt af celletype og billedopsamlingsparametre. Når du har trykket på kørslen, opretter scriptet en cellecirkulæritet og et område i forhold til tidsplottet for alle celler, der spredes.Tryk på kymographgeneratoren og analysefanen for kymograph-data. Denne analyse kræver, at inputfiler beskæres i enkeltcellebilledstakke. Når du har indsat alle indstillingerne og trykket på kørslen, opretter scriptet flere kymografier for den givne celle.Til venstre er repræsentative celler fra DMSO og CK-666 behandlinger, automatisk segmenteret ved hjælp af det medfølgende script. I modsætning til den isotropiske og meget cirkulære morfologi, som kontrolcellerne har påvist, udviser Arp2/3-hæmmede celler nedsat cirkularitet. Derudover giver de automatisk genererede kymographs tidsmæssig opløsning, der er afgørende for at forstå dynamikken i fremspring.Kontrolceller stikker vedvarende ud med lidt eller ingen tilbagetrækninger. I modsætning hertil forstyrrer Arp2/3-hæmning stabiliteten af fremspring som angivet ved stigningen i tilbagetrækningshændelser under spredning. Disse videoer skal give dig forståelse for, hvordan du korrekt frøceller, udføre farmakologiske behandlinger og forberede billeddannelse og beregningsmæssige værktøjer er nødvendige for kvantificering af cellespredning dynamik.Denne protokol kan yderligere kombineres med cytoskeleton fluorescerende billeddannelse og migration assays at identificere de molekylære spillere, der styrer celle fremspring. Tak for at se og held og lykke med dine eksperimenter.

quantitative-analysis-of-cell-edge-dynamics-during-cell-spreading

Research

JoVE Journal

Biology

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Kvantitative levende celler fluorescens-mikroskopi Analyse af Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

En kvantitativ Fitness Analyse Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Bredspredning og immunfarvning af gærkromosomer

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Høj opløsning Imaging og analyse af de enkelte Astral mikrotubulusdynamik i Spirende Gær

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Live celle Fluorescens mikroskopi for at observere væsentlige processer under mikrobielle cellevækst

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Linse-gratis Video mikroskopi for dynamiske og kvantitativ analyse af vedhængende cellekultur

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.