Diese Arbeit wurde durch Zuschüsse der NIH und ACS, einschließlich T32CA009135, CA133027, CA070723 und CA022443 finanziert. Bill Sugden ist ein American Cancer Society Research Professor.

Ein. Engineering von Zellen mit Visible Plasmide <ol><li> Verwenden Standard Restriktionsverdau oder homologe Rekombination Techniken, um eine DNA-Fragment, das etwa 250 Kopien des Lactose-Operatorsequenz (lacO) in das Plasmid zu visualisierenden einzuführen. Unsere Plasmid war ein Bacmid von etwa 170 kbp und enthalten das gesamte Genom des Kaposi-Sarkom assoziierte Herpes-Virus (KSHV) und kodierte Resistenz gegen Hygromycin 12. </li><li> Einführen LacO enthaltenden Plasmid-DNA in Säugetierzellen durch Transfektion mit Lipofectamin 2000. Wir transfizierten HeLa-Zellen und SLK in 60 mm-Schalen für 4 h mit 10 ug gereinigten Plasmid-DNA mit 25 ul Lipofectamin 2000, 0,5 ml OptiMEM, und 3,5 ml DMEM. </li><li> Ändern des Kulturmediums zu 5 ml DMEM mit 10% fötalem Rinderserum und inkubieren Zellen für 48 Stunden. </li><li> Platte die Zellen bei geringer Dichte in großen Gerichten und Kultivierung von Zellen in einem Medium mit einer Antibiotika-Selektion für die Einführung plasmid, wir ausgewählten Zellen in DMEM mit 10% fötalem Rinderserum mit 300 ug / ml Hygromycin für 3 Wochen. </li><li> Verwenden Stück sterile, Trypsin-getränkten Filterpapier Hygromycin-resistente Kolonien auf neue Kulturschalen übertragen. </li><li> Wenn transfizierten Klone wurden gezüchtet, um den Teller zu füllen, Isolieren DNA für Restriktionsverdau und Southern Blotting, um sicherzustellen, dass das Polymer der LacO Plasmid homogen ist und der erwarteten Größe. </li><li> Wird das Plasmid wurde nicht ausgeführt, um eine Verschmelzung von LacI exprimieren, infizieren geeignete Klone mit einem Retrovirus Codieren des Lactose-Repressor (lacI), fusioniert an ein rot fluoreszierendes Protein, wie LacI-tdTomato. Wir verwenden tdTomato anstatt Proteine, die näher an grün zu photoxicity, die aus mehreren Belichtungen über lange Zeiten 13 auftreten würden, zu minimieren fluoreszieren. </li><li> Sobald das LacI Fusionsprotein eingeführt wird, die Zellen Kultur in Gegenwart von 200 ug / ml Isopropyl-bD-Thiogalactosid (IPTG), um die Fusionsproteine ​​aus Bindung zu blockierenDNA. </li><li> Kultur wurden die Zellen für mindestens 3 Tage, damit Ausdruck der LacI-tdTomato Protein. </li><li> Bildschirm Klone (durch Wegwaschen der IPTG und Anzeigen Zellen wie nachstehend beschrieben) für die mit optimalen Mengen an LacI-tdTomato, die unterschiedliche Signale mit minimaler offenbaren Hintergrundwerte von ungebundenen Fusionsproteins. </li></ol> 2. Entwicklung der Imaging System Unsere Imaging-System besteht aus einem Zeiss Axiovert 200M mit einem motorisierten Bühne und Plan-Aochromat 63x/1.4 Öl-Objektiv ausgestattet. Fluoreszenzanregungslichtquelle wird durch die &quot;neutral white&quot; eines Colibri System-LED zur Verfügung gestellt. Emission wird von einem CascadeII erkannt: 1024 EMCCD Kamera. Dies ist ein gekühlter, Back-ausgedünnt Kamera mit einer Verstärkung zum Verstärken von Signalen Register; der Dunkelstrom 0,061 Elektronen pro Pixel pro Sekunde, und die Quantenausbeute größer als 0,90 ist. Das System wird durch Software gesteuert Axiovision 4,8, einschließlich der SmartExperiments Modul. Zur Detektion von tdTomazu verwenden wir eine Zeiss Filtersatz 75HE, für die 85% der übergebenen Licht kann die Fluor und 95% des emittierten Lichts zu erregen passieren die Emissions-Filter. <ol><li> Montieren eines Bildgebungssystems mit einem invertierten Mikroskop und einer EMCCD Kamera zum empfindlichen Nachweis von Signalen, eine LED-Lichtquelle für spezifische Anregungs und schnelle Schalung einer motorisierten Tisch und Fokus Arm und Software-Steuerung für automatisierte Akquisitionen von Bildern über lange Zeitintervalle mit mehrfachen z-Stacks. Das System sollte auf einem Lufttisch von Vibrationen isoliert werden sitzen. </li><li> Montieren Sie eine Bühne-top Inkubator Zellen bei 37 ° C, 5-10% CO 2 in einer feuchten Kammer halten. Genügend Zeit für das System auf eine stabile Temperatur kommen, um Bewegungsartefakte während des Experiments zu minimieren. </li><li> Verwenden Sie einen beheizten Kragen für das Ziel, das Ziel von Absaugen von Wärme aus der Probe zu verhindern. </li></ol> 3. Visualisierung markierter DNA <ol><li> Platte Zellen in einer 35 mmGlasboden Schale bei einer Dichte von weniger als 10% Konfluenz, die Paare von Zellen erlauben, in Kolonien von 8-16 Zellen ohne Überlappung unterteilen wird. Tun Sie dies mindestens 24-48 Stunden vor der Belichtung, so dass die lebensfähigen Zellen Zeit zu teilen haben. </li><li> Ein bis drei Stunden vor dem Beginn der Bildgebung, Spülen Sie die Platte 3 mal mit Kulturmedium, das sowohl selektive Drogen und IPTG fehlt. Dieses Verfahren wäscht nicht lebensfähigen Zellen und ermöglicht LacI Fusionsproteine ​​LacO Plattformen mit den Plasmiden binden. </li><li> Ersetzen des Kulturmediums mit 2 ml DMEM mit 10% fötalem Rinderserum und 25 mM HEPES. </li><li> Ersetzen Sie den oberen Teil der Kulturschale mit CultFoil gestreckt in einem Befestigungsring für einen 35-mm-Schale. Diese Abdeckung wird inhibieren Verdampfen des Kulturmediums, die Salzkonzentration über die Zeit erhöhen würde und tötet die Zellen. </li><li> Verwenden differenziellen Interferenz (DIC)-Bildgebung, die Zellen anzeigen und bestimmen die Brennebene der Ober-und Unterseite der Zellen in mehreren Sichtfelder.In jedem Feld auf eine Brennebene zu bewegen etwa 1/3 des Weges nach unten von der Oberseite der Zelle. Speichern Sie die x, y, z-Koordinaten in der Liste der gespeicherten Bühne Positionen. Anmerkung: Das Plasmid Signale möglicherweise nicht durch die Okulare, wenn Zellen durch LEDs der Wellenlänge geeignet für die LacI Fusionsprotein beleuchtet werden sichtbar. Schwache Signale können nur sichtbar mit der Verwendung des EMCCD Kamera, die Integration ermöglicht des Signals über die Zeit und damit wirkungsvoll verstärkt schwache Signale. </li><li> Im Mikroskop Steuerungssoftware, definieren eine Z-Stapel von Bildern, um an jeder Stufe gespeicherten Position durchgeführt werden. Wählen az Schrittweite von 0,35-0,50 um, die Sie zur Angleichung Nyquist in z-Richtung 14 ermöglicht. Include &quot;extra&quot; Scheiben oberhalb und unterhalb der Ebenen, in denen die Zellen befinden; diese Scheiben erlauben, den folgenden Zellbewegungen während des Zellzyklus und werden auch in der Post-Akquisition-Verarbeitung (Dekonvolution) der Bildstapel verwendet. Diese Einbeziehung wirdführen in einem Stapel von 40 bis 60 Bilder je nach der Dicke der Zellen. </li><li> Im Mikroskop Steuerungssoftware, definieren eine Zeitreihe Experiment ein Hellfeldbild des z-Stacks bei 30 min-Intervallen (zum Verfolgen Zellteilungen und Migrationen über lange Zeiten) und Fluoreszenzbilder bei 10-60 min Intervallen sammeln (zur Visualisierung Plasmiden innerhalb der Zellen). Verwenden Sie keine DIC Bildgebung während des Experiments, da es mehr Licht als Standard Hellfeldabbildung, die die Zellen ausgesetzt werden können unnötig Phototoxizität im Laufe der langen Experimenten erfordert. </li><li> Starten Sie die Software-kontrolliertes Experiment. Stellen Sie sicher, dass das System nicht gestört wird (bestoßen, ausgesetzt, um überschüssige Raumlicht, etc.) während des Experiments. </li><li> Bei Bedarf füllt die Wasser in dem Befeuchtungssystem während des Experiments. </li></ol> 4. Post-Akquisition Verarbeitung von Images <ol><li> Überprüfen Sie die Bilder für jedes z-Stack und Zeitpunkt. Ernte aus alle nicht benötigten Bereichedie Bilder auf den Computer Verarbeitungszeit in dem nächsten Schritt zu verringern. </li><li> Verwenden Sie einen Dekonvolutionsalgorithmus neu zuweisen Signalintensität der Pixel des Ursprungs in jeder z-Stapel 15. Während dieser Entfaltung auf einer theoretischen Punktstreufunktion für Ihre Bildgebungssystem basieren können, ist es vorzuziehen, die Punktstreufunktion eines bestimmten Systems durch Abbilden fluoreszierenden Mikrosphären zu messen, und diese Messung in der Entfaltungs-Algorithmus. Wir maßen die Point-Spread-Funktion unserer Imaging-System und angewandte a eingeschränkt iterativen Maximum-Likelihood-Algorithmus in AxioVision Software (Software AxioVision Beschreibung, Zeiss). </li><li> Untersuchen Sie die Bilder, um die Intensität Veränderungen und Bewegungen von Plasmiden während des Zellzyklus und die Partitionierung von Plasmiden an Tochterzellen zu verfolgen. </li></ol>

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Das hier beschriebene Verfahren kann verwendet werden, um die Plasmide in lebenden Säugetierzellen zeitlich über mehrere Generationen zu folgen. Durch die Begrenzung der Exposition gegenüber Anregungslicht, haben wir diese Techniken verwendet, um Zellen aus neu eingeteilt paarweise durch Kolonien von 16-32 Zellen folgen, die mindestens 72 Stunden und 3 Zellteilungen. Diese Versuche liefern Informationen, die nicht vom statischen Assays wie quantitative PCR, Southern Blotting und FISH erhalten werden kann. <p clas...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 11965 </td><td></td></tr><tr><td> OptiMEM </td><td> GIBCO </td><td> 31985 </td><td></td></tr><tr><td> Fötales Rinderserum </td><td> Hyclone </td><td> SH30910.03 </td><td></td></tr><tr><td> Penicillin </td><td> Sigma </td><td> P3032 </td><td></td></tr><tr><td> Streptomycinsulfat </td><td> Sigma </td><td> S9137 </td><td></td></tr><tr><td> Hygromycin </td><td> Calbiochem </td><td> 400050 </td><td> 400 ug / ml für SLK, 300 ug / ml für HeLa </td></tr><tr><td> Isopropyl-bD-Thiogalactosid (IPTG) </td><td> Roche </td><td> 10 724 815 001 </td><td> Endkonzentration 200 pg / ml </td></tr><tr&gt; <td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668-027 </td><td></td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td></td></tr><tr><td> Glass-bottom Gerichten </td><td> MatTek </td><td> P35G-1.5-10-C </td><td></td></tr><tr><td> CultFoil </td><td> Pecon </td><td> 000000-1116-08 </td><td></td></tr><tr><td> Axiovert 200M </td><td> Zeiss </td><td></td><td></td></tr><tr><td> Colibri </td><td> Zeiss </td><td> 423052-9500-000 </td><td></td></tr><tr><td> Neutral weiße LED </td><td> Zeiss </td><td> 423052-9120-000 </td><td></td></tr><tr><td> Filter Cube 75 HE </td><td> Zeiss </td><td> 489075-0000-000 </td><td></td></tr><tr><td> Plan-Apochromat 63x/1.4 Ziel </td><td> Zeiss </td><td> 440762-9904-000 </td><td></td></tr><tr><td> CascadeII: 1024 EMCCD-Kamera </td><td> Photometrics </td><td> B10C892007 </td><td></td></tr><tr><td> Tempcontrol mini </td><td> Zeiss / Pecon </td><td> 000000-1116-070 </td><td></td></tr><tr><td> Tempcontrol 37-2 digital </td><td> Zeiss / Pecon </td><td> 000000-1052-320 </td><td></td></tr><tr><td> CTI-Controller 3700 digitalen </td><td> Zeiss / Pecon </td><td> 411856-9903 </td><td></td></tr><tr><td> Ziel Heizer </td><td> Zeiss / Pecon </td><td> 440760-0000-000 </td><td></td></tr><tr><td> Stufe Heizeinsatz P </td><td> Zeiss / Pecon </td><td> 411861-9901-000 </td><td></td></tr><tr><td> Stage-top Inkubator S </td><td> Zeiss / Pecon </td><td> 411860-9902-000 </td><td></td></tr><tr><td> Luftbefeuchter-System </td><td> Zeiss / Pecon </td><td> 000000-1116-065 </td><td></td></tr></tbody></table>

monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy

Einige natürlich vorkommende Plasmide in Säuger-Zellen aufrechterhalten. Unter diesen sind Genomen von Gamma-Herpesviren, einschließlich Epstein-Barr-Virus (EBV) und Kaposi-Sarkom assoziierte Herpes-Virus (KSHV), die mehrere menschlichen Malignitäten 1-3 verursachen. Diese beiden Genomen sind in einem lizenzierten Weise jede Verwendung eines einzigen viralen Proteins und zellulären Replikationsmaschinerie repliziert und an Tochterzellen weitergegeben während der Zellteilung trotz ihrer fehlenden traditionellen Centromeren 4-8. Viel Arbeit wurde getan, um die Wiederholungen dieser Plasmid Genome Charakterisierung mit Methoden wie Southern-Blot-und Fluoreszenz-in-situ-Hybridisierung (FISH). Diese Methoden sind beschränkt, though. Quantitative PCR und Southern-Blots geben Auskunft über die durchschnittliche Anzahl von Plasmiden pro Zelle in einer Population von Zellen. FISH ist ein Single-Zell-Assay, der sowohl die durchschnittliche Anzahl und die Verteilung von Plasmid enthüllt<html> s pro Zelle in der Population von Zellen, jedoch statisch ist, so dass keine Informationen über die Eltern oder Nachkommen des untersuchten Zelle. Hier beschreiben wir ein Verfahren zur Visualisierung von Plasmiden in lebenden Zellen. Dieses Verfahren basiert auf der Bindung eines fluoreszenzmarkierten Lactose Repressorproteins an mehrere Stellen des Plasmids von Interesse 9 basiert. Die DNA von Interesse wird ausgeführt, um etwa 250 Tandemwiederholungen des Lactose-Operator (LacO)-Sequenz umfassen. LacO speziell vom Lactose-Repressor-Protein (LacI), die mit einem fluoreszierenden Protein fusioniert werden kann, gebunden. Das Fusionsprotein kann entweder aus dem konstruierten Plasmids oder eingeführt durch einen retroviralen Vektor exprimiert werden. Auf diese Weise werden die DNA-Moleküle fluoreszierend markierten und damit sichtbar werden über Fluoreszenzmikroskopie. Das Fusionsprotein wird die Bindung der Plasmid-DNA durch Kultivieren von Zellen in der Gegenwart von IPTG blockiert, bis die Plasmide bereit, zu betrachten sind. nhalt &quot;&gt; Dieses System ermöglicht die Plasmide in lebenden Zellen über mehrere Generationen, offenbart Eigenschaften ihrer Synthese und Partitionierung Tochterzellen überwacht werden. Ideal Zellen haftende, leicht transfiziert und haben große Kerne. Diese Technik wurde verwendet, um festzustellen, dass 84% der EBV-abgeleitete Plasmide jede Generation synthetisiert und 88% des neu synthetisierten Plasmide Partition getreu Tochterzellen in HeLa-Zellen. Paare dieser EBV Plasmide wurden als zu angebunden werden oder damit verbunden Schwesterchromatiden nach ihrer Synthese in der S- Phase bis sie gesehen wurden, wie die Schwesterchromatiden in Anaphase 10 getrennt voneinander zu trennen. Die Methode wird derzeit verwendet, um die Replikation von KSHV Genome in HeLa-Zellen und SLK Zellen zu untersuchen. HeLa-Zellen werden immortalisierten humanen Epithelzellen und SLK Zellen immortalisierten humanen Endothelzellen Zellen. Obwohl SLK Zellen wurden ursprünglich aus einem KSHV Läsion abgeleitet, weder die HeLa noch SLK Zelllinie natürlich harbors KSHV Genome 11. Neben dem Studium Virusreplikation kann diese Visualisierungstechnik verwendet, um die Wirkungen der Zugabe, Entfernen oder Mutation der DNA-Sequenz verschiedenen Elemente auf Synthesegas-, Lokalisierungs-und Partitionierung von anderen rekombinanten Plasmid-DNAs zu untersuchen.

<html> Maximalintensität Vorsprünge von z-Stacks in einem repräsentativen Experiment erworbenen sind in Abbildung 3 dargestellt. Typische Plasmid Signale vorhanden sind in 3-8 Scheiben des Z-Stapel, je nachdem, ob sie für Einfachbindungen oder Clustern von Plasmiden. Im Fall von EBV-und KSHV abgeleitete Plasmide, bewegen sich die Signale innerhalb der Zelle langsam, bis die Zelle gelangt Mitose. Die Zellen selbst wandern über den Verlauf des Experiments. Sie werden auch sphärische und lösen sich v...

Watch this Scientific Journal Video about Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy at JoVE.com

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Verfahren zur Beobachtung der einzelnen DNA-Molekülen in lebende Zellen beschrieben. Die Technik basiert auf der Bindung eines fluoreszenzmarkierten Proteins an lac-Repressor-Bindungsstellen in die DNA von Interesse entwickelt basiert. Dieses Verfahren kann gut auf rekombinanten DNAs in lebende Zellen im Laufe der Zeit zu folgen.

Überwachung Plasmid-Replikation in lebenden Säugerzellen über mehrere Generationen durch Fluoreszenz-Mikroskopie

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

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11.7K Aufrufe.  University of Wisconsin - Madison. Verfahren zur Beobachtung der einzelnen DNA-Molekülen in lebende Zellen beschrieben. Die Technik basiert auf der Bindung eines fluoreszenzmarkierten Proteins an lac-Repressor-Bindungsstellen in die DNA von Interesse entwickelt basiert. Dieses Verfahren kann gut auf rekombinanten DNAs in lebende Zellen im Laufe der Zeit zu folgen.

Serie: Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Langfristige Imaging Säugetierzellen mit Wide-Field-Mikroskopie

Forschung

Biologie

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizing Einzel-Molekül-DNA-Replikation mit Fluoreszenz-Mikroskopie

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zeitraffer-Imaging der Mitose Nach siRNA-Transfektion

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Eine Fluoreszenz-Mikroskopie Assay zur Überwachung Mitophagy in der Hefe Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluoreszenz-basierte Messung von Calcium-Shop-Eintrag in lebenden Zellen: aus kultivierten Krebszelle zu Skelettmuskelfaser

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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.

Quantitative Live Cell Fluoreszenz-Mikroskopie Analyse von 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 ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET-Mikroskopie zur Echtzeit-Überwachung der Signalisierung Events in lebenden Zellen mit unimolekularen Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Der Nachweis von intrazellulären Genexpression in lebenden Zellen der Maus, Mensch und Schwein Origin Mit Fluoreszenz-markierter Nanopartikel

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...