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 arbeidet ble støttet av Connaught Fund New Investigator Award til S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (tilskudd RGPIN-2015-05114 og RGPIN-2020-05881), University of Manchester og University of Toronto Joint Research Fund, og University of Toronto XSeed Program.

1. Celle såing
MERK: Den beskrevne cellespredningsprotokollen ble utført ved hjelp av musens embryonale fibroblaster (MEFer) som uttrykker PH-Akt-GFP (en fluorescerende markør for PIP3/PI(3,4)P2). Denne cellelinjen ble generert ved genomisk integrering av en uttrykkskonstruksjon for PH-Akt-GFP (Addgene #21218) ved CRISPR-mediert genredigering. Imidlertid kan andre fluorescerende markører som uttrykkes forbigående eller integrert i genomet også brukes i denne analysen....

<ol>
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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. 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Den beskrevne cellespredningsanalysen muliggjør kontinuerlig sporing av morfologiske endringer(f.eks. cellestørrelse og -form) og cellekantbevegelser (dvs. fremspringshastighet og tilbaketrekkingsfrekvens), som mangler i de fleste cellespredningsprotokoller19,24. Mens ofte brukte sluttpunktcellespredningsanalyser tillater bestemmelse av cellespredningshastighet, klarer ikke disse analysene å løse den tidsmessige dynamikken i cellekantbevegel...

Lamellipodiale fremspring er fremtredende cytoskeletale strukturer dannet på forsiden av en migrerende celle. I lamellipodia skaper polymerisering av aktin ved hjelp av Arp2/3-komplekset og formins et raskt voksende forgrenet aktinnettverk som skyver mot plasmamembranen1,2. Trykkkraften som genereres av aktinnettverket, driver fysisk cellen fremover1,3,4,5. Uttømming av Arp2/3-komplekset eller forstyrrelse av signalveier som er avgjørende for lamellipodiale fremspring, svekker ofte cellemigrasjon6, 7. Selv om migrasjon av lamellipodia-mangelfulle celler også har blitt rapportert8,9, er betydningen av lamellipodia i cellemigrasjon tydelig som uttømming av denne fremspringende strukturen perturberer cellens evne til å bevege seg gjennom komplekse biologiske mikromiljøet6,10.
En stor hindring for å forstå reguleringen av lamellipodia i migrerende celler er den naturlige variasjonen i lamellipodial fremspring kinetikk, størrelse og form11,12,13,14. Videre har nyere studier vist at lamellipodia viser kompleks fremspringende oppførsel, inkludert svingende, periodiske og akselererende fremspring14,15. Sammenlignet med den svært variable lamellipodia av migrerende celler6,16, er lamellipodia dannet under cellespredning mer ensartet12. Siden den fremspringende aktiviteten med å spre og migrere celler er drevet av identiske makromolekylære samlinger, som inkluderer et forgrenet aktinnettverk, sammentrekningsformede actomyosinbunter og integrinbaserte cellematriseadhesjoner17,18, har spredningsceller blitt mye brukt som modell for å undersøke reguleringen av lamellipodiadynamikk.
Cellespredning er en dynamisk mekanokjemisk prosess der en celle i suspensjon først holder seg til et substrat gjennom integrinbaserte adhesjoner17,19,20 og deretter sprer seg ved å utvide aktinbaserte fremspring21,22,23. Under spredningsfasen stikker lamellipodia ut fra cellekroppen isotropisk og vedvarende med liten eller ingen tilbaketrekning eller stalling12. De mest brukte cellespredningsprotokollene er endepunktanalyser, der spredning av celler er faste på forskjellige tidspunkter etter plating19,24. Disse analysene, selv om de er raske og enkle, er begrenset i sin diagnostiske kraft til å oppdage endringer i de dynamiske egenskapene til lamellipodia. For å bestemme de molekylære mekanismene som kontrollerer lamellipodiadynamikken, var Sheetz-gruppen en pioner innen bruk av kvantitativ analyse av levende spredningsceller og avdekket mange grunnleggende egenskaper ved cellekantprotrudering11,12,22. Disse studiene har vist at live-celle spredningsanalysen er en robust og kraftig teknikk i verktøykassen til et cellebiologisk laboratorium. Til tross for det er et detaljert protokoll- og åpen kildekode-beregningsverktøy for en live-celle spredningsanalyse for tiden utilgjengelig for cellebiologisamfunnet. For dette formål skisserer protokollen vår prosedyrene for avbildning av direkte spredningsceller og gir et automatisert bildeanalyseverktøy. For å validere denne metoden brukte vi Arp2/3-hemming som eksperimentell behandling og viste at hemming av funksjonen til Arp2/3-komplekset ikke arresterte cellespredning, men forårsaket en betydelig reduksjon i celleutstikkhastigheten, samt stabiliteten til cellekantutstikk, noe som ga opphav til hakkede cellekanter. Disse dataene viser at kombinasjonen av levende celleavbildning og automatisert bildeanalyse er et nyttig verktøy for å analysere cellekantdynamikk og identifisere molekylære komponenter som 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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>1-Well Chamlide CMS for 22 mm x 22 mm Coverslip</td>
			<td>Quorum Technologies</td>
			<td>CM-S22-1</td>
			<td></td>
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		<tr>
			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
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			<td>50 mL Centrifuge Tube, Transparent, Plug Seal</td>
			<td>Nest</td>
			<td>602002</td>
			<td></td>
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		<tr>
			<td>6.0 cm Cell Culture Dishes Treated for Increased Cell Attachment, Sterile</td>
			<td>VWR</td>
			<td>10861-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Arp2/3 Complex Inhibitor I, CK-666</td>
			<td>Millipore Sigma</td>
			<td>182515</td>
			<td></td>
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			<td>Camera, Prime 95B-25MM</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
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			<td>Dimethyl Sulfoxide, Sterile</td>
			<td>BioShop</td>
			<td>DMS666</td>
			<td></td>
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		<tr>
			<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>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, No Phenol Red</td>
			<td>Gibco</td>
			<td>11039021</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS, 1X</td>
			<td>Wisent Bioproducts</td>
			<td>311-425 CL</td>
			<td></td>
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			<td>Fetal Bovine Serum</td>
			<td>Wisent Bioproducts</td>
			<td>080-110</td>
			<td></td>
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			<td>Fiji Software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
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			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
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			<td>Human Plasma Fibronectin Purified Protein 1 mg</td>
			<td>Millipore Sigma</td>
			<td>FC010</td>
			<td></td>
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			<td>Immersion Oil</td>
			<td>Cargille</td>
			<td>16241</td>
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			<td>L-Glutamine Solution (200 mM)</td>
			<td>Wisent Bioproducts</td>
			<td>609-065-EL</td>
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			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140050</td>
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			<td>Micro Cover Glasses, Square, No. 11/2 22 x 22 mm</td>
			<td>VWR</td>
			<td>CA48366-227-1</td>
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			<td>Microscope Body, Eclipse Ti2-E</td>
			<td>Nikon</td>
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			<td>Objective, CFI Plan Apo Lambda 60X Oil</td>
			<td>Nikon</td>
			<td>MRD01605</td>
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			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</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>
			<td></td>
			<td></td>
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			<td>Stage top incubator</td>
			<td>Tokai Hit</td>
			<td></td>
			<td></td>
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			<td>Statistics Software, Prism</td>
			<td>GraphPad</td>
			<td></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

Celleoppslag er en dynamisk prosess der en celle som er suspendert i medier, knyttes til et underlag og flater seg ut fra en avrundet til en tynn og spredt form. Etter celle-substratet danner cellen et tynt ark med lamellipodia som kommer fra cellekroppen. I lamellipodia polymeriserer kulefikale aktin (G-aktin) monomerer til et tett filamentøst aktin (F-aktin) maskearbeid som skyver mot plasmamembranen, og gir dermed de mekaniske kreftene som kreves for at cellen skal spre seg. Spesielt er de molekylære spillerne som kontrollerer aktinpolymeriseringen i lamellipodia avgjørende for mange andre cellulære prosesser, for eksempel cellemigrasjon og endokytose. 
Siden spredning av celler danner kontinuerlig lamellipodia som spenner over hele celleperiphery og vedvarende ekspanderer utover, har cellespredningsanalyser blitt et effektivt verktøy for å vurdere kinetikken til lamellipodiale fremspring. Selv om flere tekniske implementeringer av cellespredningsanalysen er utviklet, mangler det for tiden en detaljert beskrivelse av arbeidsflyten, som vil inkludere både en trinnvis protokoll og beregningsverktøy for dataanalyse. Her beskriver vi de eksperimentelle prosedyrene for cellespredningsanalysen og presenterer et åpen kildekodeverktøy for kvantitativ og objektiv analyse av cellekantdynamikk under spredning. Kombinert med farmakologiske manipulasjoner og/eller gen-silencing teknikker, er denne protokollen egnet til en storskala skjerm av molekylære spillere som regulerer lamellipodiale fremspring.

Protokollen ovenfor beskriver de eksperimentelle prosedyrene for live-celleavbildning av spredningsceller og et beregningsverktøy for kvantitativ analyse av cellespredningsdynamikk. Beregningsverktøyet kan brukes i et lav- eller høygjennomstrømningsformat for å identifisere molekylære aktører som regulerer aktinpolymeriseringsmaskineriet i celleforkanten.
Den skjematiske representasjonen av de eksperimentelle prosedyrene er avbildet i figur 1. Cellesprednin...

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 protokollen presenterer vi eksperimentelle prosedyrer for en cellespredningsanalyse som er basert på levende cellemikroskopi. Vi tilbyr et beregningsverktøy med åpen kildekode for objektiv segmentering av fluorescerende merkede celler og kvantitativ analyse av lamellipodiadynamikk under cellespredning.

Kvantitativ analyse av cellekantdynamikk 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 beskrevet i denne videoen vil definere de eksperimentelle prosedyrene som kreves for avbildning av spredningsceller i livsstørrelse og bruk av et beregningsverktøy som kvantifiserer celler som sprer dynamikk på en objektiv og automatisert måte. Cellespredningsressursen gjør det mulig å spore cellekantbevegelser kontinuerlig og morfologiske endringer under cellespredning, som er en funksjon som mangler i de fleste eksisterende cellespredningsanalyser. I tillegg implementerer denne protokollen bruk av automatisert databehandling og analyse, og gir informasjon om cellesirkularitet, areal- og fremsprings tilbaketrekkingssykluser for spredning av celler.Slik automatisert behandling reduserer skjevheter i dataanalyse og gir en robust metode for å analysere cellespredningsdynamikk for et stort antall celler. Når den kombineres med narkotikabehandlinger eller genvitenskap og teknikker, er denne protokollen egnet til store hastigheter av molekylære spillere som regulerer celleutstikk. For den gitte protokollen, Cellene som brukes er mus embryonale fibroblaster som genetisk koder PH-Akt-GFP, noe som muliggjør fluorescerende merking av plasmamembranen.Før begynnelsen av cellen sprer analyse, kultur en tallerken med celler til 90% samløp. Når cellene har oppnådd riktig samløp, flamme en 22 x 22 millimeter deksel slip og plassere den i en 35 millimeter celle kulturrett. Belegge dekselslippet med fibronektin som har blitt fortynnet i PBS til en konsentrasjon på 2,5 mikrogram per milliliter.Plasser retten med dekselet gli inn i en 37 graders inkubator i en time. Etter en time aspirerer du fibronectinet og sørger for ikke å berøre dekselslippet med pipettespissen. Vask fatet med PBS ved å pipetter forsiktig rundt dekselet slip to til tre ganger.For cellesåing begynner du med å aspirere cellekulturmediene fra parabolen av celler. Deretter vasker du parabolen med varm PBS. Tilsett 650 mikroliter trypsin EDTA i parabolen og vipp parabolen jevnt for å fordele enzymet jevnt.Plasser parabolen med trypsin i inkubatoren i ett minutt. Etter inkubasjon må du først legge til 10 milliliter cellekulturmedier i et sentrifugerør. Deretter legger du raskt til ytterligere 10 milliliter medier i parabolen for å slukke trypsin.For å fortynne cellene som skal frøs på dekselslippet, pipette en milliliter av parabolens innhold inn i sentrifugerøret. Fra røret, pipette ca 500 til 1000 mikroliter fortynnede celler inn i parabolen med dekselslippet. Forsikre deg om at dekselslippet er på 10% samløp eller 50.000 celler per milliliter og juster volumet av fortynnede celler etter behov.Hensikten med disse cellene er å etablere et fokusplan under bildeanskaffelse. Med de resterende cellene i parabolen, passerer du en femtedel av cellene i en liten tallerken per behandling. Dette vil være cellene som vil bli analysert for spredning av dynamikk.Plasser passasjerettene og dekselets glidefat i en inkubator i åtte til 24 timer. For å teste betydningen av Arp2/3 for cellespredning, legg først fem milliliter cellekulturmedier inn i en kontroll og et behandlingssentrifugerør. Deretter legger du til 20 milliliter dmem som mangler fenolrød i hvert av to større sentrifugerør.Pipette enten stoffet CK-666 som er en hemmer av Arp2/3-komplekset eller den kontrollerte behandlingen som DMSO i de små og store rørene. For å begynne farmakologisk behandling av cellene, fjern passasjerettene som inkuberer over natten. Aspirer cellekulturmediene fra alle rettene og vask oppvasken med varm PBS.Ta de små rørene som inneholder enten CK-666 eller kontrollsupplerte medier og legg til innholdet i det aktuelle røret i hver av passasjerettene. Merk hver av rettene med riktig legemiddelbehandling, og legg deretter oppvasken tilbake i inkubatoren i en ekstra time Etter en times inkubasjon aspirerer du stoffet supplert media fra hver av rettene. Deretter legger du til varm PBS til alle rettene for å fjerne alle de gjenværende fenolrøde mediene grundig.Tilsett 230 mikroliter trypsin EDTA og legg oppvasken i en inkubator i ett minutt. Fjern de trypsiniserte rettene fra inkubatoren og fortsett å legge til fem milliliter av stoffet supplert dmem som mangler fenolrød i et rør utpekt som rør B.Legg deretter ytterligere fem milliliter av samme medier inn i den aktuelle parabolen for å slukke trypsin. Pipette media opp og ned flere ganger for å løsne alle cellene fra parabolen.Overfør alt innholdet i parabolen til et annet rør som allerede er utpekt som rør A.In for å forberede en cellefortynning som passer for avbildning, overfør en milliliter celler fra rør A til rør B.Gjenta alle fortynningstrinnene for hver behandling. Plasser alle rørene i inkubatoren i 45 minutter for å la cellene komme seg etter trypsinisering. Sørg for at alle deler av et cellemagnetisk kammer som har plass til en 22 x 22 millimeter dekselsglidning, er grundig rengjort før bruk.Fjern fatet med dekselslippet fra inkubatoren. Aspirer cellekulturmediene og vask dekselslippet med varm PBS. Fjern dekselslippet med et par tang og legg forsiktig dekselslippet på bunnplaten på det magnetiske kammeret.Deretter plukker du opp silikonpakningen og legger den på toppen av dekselslippet. Fest hoveddelen av det magnetiske kammeret på bunnplaten. Tilsett en milliliter av dmemen som mangler fenolrød som tilsvarer den aktuelle behandlingen til det magnetiske kammeret.Ta et lofritt vev og forsiktig dab kabinettet mellom hoveddelen og bunnplaten for å se etter lekkasjer. For å fullføre det magnetiske kammeret, senk det gjennomsiktige dekselet på hoveddelen. Tørk til slutt bunnen av dekselslippet med et vev sprayet med vann og en annen sprayet med 70% etanol.Forvarm scenen topp inkubator av et konfokalt mikroskop til 37 grader. Plasser det magnetiske kammeret på toppen av målet. Før du kjøper spredningsdynamikk, sett fokus på de allerede polariserte cellene i den grønne fluorescerende kanalen.Dette sikrer at spredningscellene er i fokus når de festes til dekselslippet. Fjern det gjennomsiktige dekselet til magnetkammeret og pipetten 500 mikroliter fra rør B som ble fjernet fra inkubatoren. Plasser det gjennomsiktige dekselet på toppen igjen.For å identifisere celler som er ideelle for cellespredningsanalyse, søk etter grønne glorier som representerer celler som ennå ikke er festet til omslagsseddelen eller er i de tidligste stadiene av vedlegget. Hent bilder og lagre filene. Etter å ha fullført bildeanskaffelsen av cellespredning, begynner du med å kjøre en kompatibel Python IDE som Spyder.For beregningsanalyse av cellespredningsdynamikk, finn og åpne cellen som sprer GUI-filen som er gitt i de relevante skriptpakkene. Trykk på kjør-knappen som åpner analyse-GUI-panelet i bakgrunnen. GUI huser alle innstillingene som trengs for analyse.Hvis du vil analysere celleområde og sirkularitet, skriver du inn anskaffelsesfilen og de nødvendige innstillingene i kategorien celleoppslagsområde. Justeringer må gjøres avhengig av celletype og bildeanskaffelsesparametere. Etter å ha trykket på kjøringen, vil skriptet opprette en celle sirkularitet og areal kontra tidsplott for alle spredende celler.For kymografdata trykker du på kymografgeneratoren og analysefanen. Denne analysen krever at inndatafiler beskjæres enkeltcellede bildestakker. Etter å ha satt inn alle innstillingene og trykket på kjør, vil skriptet opprette flere kymografier for den gitte cellen.Til venstre er representative celler fra DMSO- og CK-666-behandlingene, automatisk segmentert ved hjelp av det medfølgende skriptet. I motsetning til den isotropiske og svært sirkulære morfologien demonstrert av kontrollceller, viser Arp2/3-hemmet celler redusert sirkularitet. I tillegg gir de automatisk genererte kymografiene timelig oppløsning som er avgjørende for å forstå dynamikken i fremspring.Kontrollceller stikker ut vedvarende med lite eller ingen tilbaketrekninger. Til sammenligning forstyrrer Arp2/3-hemming stabiliteten til fremspringene som indikert av økningen i tilbaketrekningshendelser gjennom spredning. Denne videoene skal gi deg forståelse for hvordan du skal frøceller riktig, utføre farmakologiske behandlinger og forberede avbildnings- og beregningsverktøy som er nødvendige for kvantifisering av cellespredningsdynamikk.Denne protokollen kan kombineres ytterligere med cytoskeleton fluorescerende avbildnings- og migrasjonsanalyser for å identifisere molekylære aktører som styrer celleutstikk. Takk for at du så på og lykke til med eksperimentene dine.

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 Live-Cell fluorescens-mikroskopi Analyse av Fission gjær

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 fitnessanalyse 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.

Surface Sprer og Farging av gjær Kromosomer

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.

Høyoppløselig avbildning og analyse av enkelt Astral mikrotubulidynamikk i Budding gjær

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 å observere viktige prosesser under mikrobiell cellevekst

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.

Objektivet-gratis Video mikroskopi for dynamisk og kvantitativ analyse av tilhenger 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.