Wir danken Rev. Dr. Ralph Bohlmann und Rev. Dr. James Voelz für die Annahme, die das Wort &quot;Anastasis&quot;, um Umkehr der Apoptose zu beschreiben; Douglas R. Green für HeLa-Zellen, die Cytochrom-c--GFP; Charles M. Rudin und Eric E. Gardner für H446-Zellen; Heather Lamb für die Unterstützung bei Cartoon-Zeichnung auf dem Video; Yee Hui Yeo für wertvolle Diskussion des Manuskripts. Diese Arbeit wurde von einem Sir Edward Youde Memorial Fellowship (HLT), die Dr. Walter Szeto Memorial Scholarship (HLT), Fulbright-Stipendium 007-2009 (HLT), Life Science Research Foundation Fellowship (HLT) unterstützt wird, gewährt NIH NS037402 (JMH) und NS083373 (JMH) und University Grants Committee der Hong Kong AoE / B-07/99 (MCF). Ho Lam Tang ist ein Shurl und Kay Curci Foundation Fellow der Life Sciences Research Foundation.

The authors have nothing to disclose.

Anastasis bezieht sich auf das Phänomen, bei dem Zellen, Zelltod aktiviert haben danach rückgängig den Sterbeprozess und zu überleben. Hier haben wir gezeigt, dass die Abbildung lebender Zellen verwendet werden, um zu bestätigen, dass die gleichen Einzelzellen in der Tat kann Apoptose-Prozess in einer späten Phase umkehren, und dann weiter überlebende und Wiedergabe werden. Unsere Protokolle beschreiben mehrere optimierte Zelltyp-spezifischen Behandlungsbedingungen, Apoptose zu induzieren und lassen einen großen...

Apoptose (griechisch für &quot;fallen in den Tod&quot;) wird im Allgemeinen angenommen, dass ein einseitiger Prozess endet in Zelle Selbstmord 1-7 sein. Genetische Störung der pro-Tod Gene Ergebnisse in der Überlebensrate von zusätzlichen Zellen, die sonst in ganzen Tieren sterben würde, einschließlich Zellen, die bereits die Apoptoseweg 8,9 eingeleitet haben. Auch genetische Manipulationen ermöglichen gesunden Säugetierzellen, die künstlich Anzeige &quot;essen mich&quot; Signale oder die Haftfähigkeit zu verlieren, ihre extrazellulären Matrix zum Tod durch Ganzzell-Phagozytose oder entosis bzw. 10,11 entkommen. Jedoch haben wir und andere gezeigt, dass ohne genetische Manipulation normalen gesunden Säugetierzellen und Zelllinien, können auch aus den frühen Stadien der Apoptose 12-15 erholen. Mit Werkzeugen, einzelne Zellen zu verfolgen, haben wir weiter gezeigt, sich nach späten Stadien der Apoptose 12,13, nachdem Zellen wichtige Kontrollpunkte, dass typische gebenly markieren den &quot;point of no return&quot; 2-6. Diese Checkpoints Stufe Apoptose spät schließen mitochondrialen Freisetzung von Cytochrom c, Aktivierung von Caspasen, eine Kernfragmentierung und Bildung von apoptotischen Körpern. Wir nahm eine griechische gesetztes Wort &quot;Anastasis&quot;, das bedeutet &quot;stieg auf Leben&quot;, um diese Umkehrung der Apoptose am Rande der Zelltod 2-6 beschreiben. Es sei denn, die gesamte Sterbe-Recovery-Prozess wird von lebenden Zellen beobachtet wird, wird es eine Herausforderung, Zellen, die anastasis aus Zellen, die nie apoptotischen Ereignisse erlebt unterzogen haben zu unterscheiden. Die jahrzehntelange Arbeit haben gezeigt, dass die morphologischen Merkmale der Zelltod durch Apoptose durch evolutionär konservierte biochemischen und molekularen Ereignisse 16-19 angetrieben. Diese Veranstaltungen fördern die Selbstzerstörung der Zellen, Entwicklungs- und homöostatischen Prozesse in einzelligen und mehrzelligen Organismen durch die Beseitigung beschädigter oder d zu regelnangerous Zellen 16-19. Während apoptotischen Zellen können leicht durch standardisierte morphologischen, biochemischen und molekularen Manifestationen Apoptose 1,5,6,16,20 unterschieden werden, gibt es bisher keine bekannte Marker spezifisch anastasis 12,13. Wichtig ist, dass Zellen, die anastasis laufen haben scheinen normale gesunde Zellen und Zellen, die gerade beginnen Umkehr Apoptose erscheinen als apoptotische sterbenden Zellen 12,13 sein. Damit neue Werkzeuge erforderlich sind, um mit Gewissheit, dass ein bestimmter überlebenden Zellen zuvor erlebt aktiven apoptotischen Prozessen abzuschließen. Apoptose ist im Allgemeinen als eine irreversible Kaskade angenommen, weil es eine schnelle und massive Zerstörungsprozess. Man könnte zwar Minuten bis Tage dauern für einige Zellen, die Apoptose auslösen, sobald Mitochondrien apoptogene Faktoren wie Cytochrom c in das Cytosol freigesetzt 21,22 kann Caspasen innerhalb von 5 Minuten 23,24 aktiviert werden, gefolgt von cytoplasmatischen undKernkondensation innerhalb von 10 Minuten 25 bis 27, und Zelltod kurz danach 25-27. Aktivierte Caspasen dirigieren Apoptose durch Spaltung und Inaktivierung wichtigsten strukturellen und funktionellen Komponenten für die Zwecke der zellulären Abbruch 2,28, wie die Endonuklease-Inhibitor DFF45 / ICAD 29,30. Caspasen aktiviert auch pro-apoptotischen Faktoren wie BCL-2-Familienmitglied BID, die Mitochondrien transloziert mitochondriale Freisetzung von Cytochrom c 31,32 fördern. Caspase-Aktivität führt auch zu Zelloberflächenexposition von Phosphatidylserin als &quot;essen mich&quot; Signal für die Förderung der Phagozytose von sterbenden Zellen durch Makrophagen oder Nachbarzellen durch Phagozytose 33. Außerdem apoptotische Vorgänge machen Mitochondrien dysfunktional, Störung zellulärer Bioenergetik und Stoffwechsel 34,35,36. So scheint Erholung von solchen Zerstörung intuitiv unwahrscheinlich. Entgegen der ursprünglichen erwartentionen können Zellen die Apoptose Prozess noch in einem späten Stadium umzukehren. Durch die kontinuierliche Überwachung das Schicksal der sterbenden Zellen in Kultur, beobachteten wir die Reversibilität der Bühne Apoptose spät in einem Spektrum von primären Zellen und Zelllinien 12,13. Die Entfernung der Tod Reizes Erholung von den offenkundigen Eigenschaften der Apoptose, wie mitochondriale Fragmentierung, Chromatin-Kondensation, DNA-Schäden, Plasmamembran Blasenbildung, Zelloberflächenexposition von Phosphatidylserin, Freigabe der mitochondrialen Cytochrom C, Caspase-Aktivierung, Kernfragmentierung, Zellschrumpfung, und Bildung von apoptotischen Körpern. Diese Beobachtungen werfen unbeantwortete Fragen zu den Funktionen, Folgen und Mechanismen der Rekonvaleszenz. Um diese Fragen anzugehen, ist eine Voraussetzung, um zuverlässig zu identifizieren Zellen, anastasis unterzogen wurden. Hier beschreiben wir Live-Mikroskopieverfahren und eine Caspase Biosensor zum Nachweis von Zellen, die zuvor rückgängig gemacht haben, spät Apoptose und then überlebt.

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strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis

Anastasis (griechisch für &quot;Rising to life&quot;) bezieht sich auf die Wiederherstellung von sterbenden Zellen. Bevor diese Zellen zu gewinnen, sie durch wichtige Kontrollpunkte der Apoptose, einschließlich mitochondriale Fragmentierung, Freigabe der mitochondrialen Cytochrom c in das Zytosol, Aktivierung von Caspasen, Chromatin-Kondensation, DNA-Schäden, Kernfragmentierung, Plasmamembran Blasenbildung, Zellschrumpfung, Zelloberflächenexposition bestanden haben Phosphatidylserin und Bildung von apoptotischen Körpern. Anastasis kann auftreten, wenn apoptotische Reize vor dem Tod entfernt, wodurch sterbenden Zellen zur Apoptose und möglicherweise anderen Todesmechanismen umzukehren. Daher erscheint anastasis physiologischen Heilungsprozesse, die auch beschädigte Zellen zu erhalten könnte unangemessen einzubeziehen. Die Funktionen und Mechanismen der Rekonvaleszenz sind noch unklar, zum Teil durch die begrenzte Tools zur Erkennung Ereignissen der Vergangenheit nach der Wiederherstellung der scheinbar gesunden Zellen behindert. Strategien zur Anasta erkennensis werden Studien über die physiologischen Mechanismen, die Gefahren von Untoten Zellen in Krankheitspathologie und potenziellen Therapeutika ermöglichen anastasis modulieren. Hier beschreiben wir effektive Strategien mit Lebendzellmikroskopie und eine Säugetier Caspase Biosensor zur Identifikation und Verfolgung anastasis in Säugerzellen.

Um die Auflösung der Apoptose zu untersuchen, werden Gewebekulturzellen zunächst auf einen Tod Reiz ausgesetzt, die Apoptose auslösen. Wenn die Zellen anzuzeigen Kennzeichen der Apoptose wird frisches Kulturmedium dann angewendet, um abzuwaschen den Reiz und bebrüten die sterbenden Zellen der Besserung (1A) zu ermöglichen. Hier ist die entscheidende Frage angesprochen, wie weit einzelne sterben kultivierten Zellen gegenüber Apoptose Fortschritt und noch anastasis eingehen können. Diese Frage läs...

Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Strategien für die Spurhaltung Anastasis, einer Zelle Überleben Phänomen, das Apoptose Kehrt

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

strategies-for-tracking-anastasis-cell-survival-phenomenon-that

Research

JoVE Journal

Biology

17.9K Aufrufe.  Johns Hopkins University Bloomberg School of Public Health. The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis. 

Serie: Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Überwachung dynamischen Veränderungen in der mitochondrialen Kalzium während der Apoptose mit einem genetisch kodierten Kalzium-Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Forschung

Biologie

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Geändert Annexin V / Propidiumiodid Apoptose Assay für eine genaue Beurteilung des Zelltods

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Die Aktivierung der Apoptose durch cytoplasmatische Mikroinjektion von Cytochrom C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Tracking Morphogenetic Tissue Verformungen in der frühen Hühnerembryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Formulierungen für die Gefriertrocknung von Bakterien und deren Einfluss auf Zellebensdauer

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimiert Färbung und Proliferation Modeling-Methoden für die Zellteilung Überwachung mit Cell-Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Handy-Tracking Mit Photoconvertible Proteine ​​während der Zebrafisch-Entwicklung

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Die Tomate / GFP-FLP / FRT Verfahren zur Live-Imaging von Mosaic Erwachsene<em&gt; Drosophila</em&gt; Sehzellen

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

A Guide to Modern Quantitative Fluoreszenz Western Blot mit Strategien zur Fehlersuche

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Bewertung der Selective mRNA Translation in Säugerzellen durch Polysomen Profilieren

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...