1. Vorbereitung der Lipide <ol><li> Um Proteine ​​an die obere Flugblatt einer Doppelschicht Link verwenden wir Ni 2 + Komplexbildner Lipide, die rekombinante Proteine ​​mit Poly-Histidin-tags zu verankern. Bestimmte Zelltypen können nicht speziell an Ni 2 +-beschichtete Doppelschichten, aber T-Zellen in der Regel nicht, so dass diese Methode optimal für das Studium T-Zell-Aktivierung. </li><li> Die Lipid-Doppelschicht-Lösung wird durch Herstellen einer Lösung von 20% Ni 2 + gemacht chelatisierenden Lipid-und 80% Phosphatidylcholin in die entsprechenden Beträge in ein Glasrohr mit 1-2 ml Chloroform-Methanol. <ol><li> Das Lösungsmittel wird unter einem Strom von Stickstoffgas in einem warmen (30-37 ° C) Wasserbad. Dies dauert in der Regel etwa 15 Minuten. </li><li> Entfernen Sie alle verbleibenden Lösungsmittels im Hochvakuum mit einem Lyophilisator für 90 Minuten. </li></ol></li><li> Lösen Sie die Lipide in 2% N-Octyl-Glucosid Reinigungsmittel in Tris-Saline-Puffer, der eine Lösung von gemischt Waschmittel / Phospholipid-Micellen erzeugt. Die endgültige Phospholipidkonzentration sollte 0,4 mm betragen. Zur Bildung Liposomen, dialysieren diese Lösung gegen 3 Änderungen der Tris-Saline zu Wasch-und Form-Liposomen zu entfernen. Wir in der Regel mit einem Volumen Arbeit in der Größenordnung von 1 ml mit 6 mm Durchmesser Spectra / Por # 2 Schläuche mit einem Cut-off um 10 kDa. Wir sind darauf bedacht, jede Luftblase s auszuschließen beim Spannen des Rohres. Wir in der Regel steril-Filter diese Lösung und nicht die Dialyse unter sauberen Bedingungen, die Handhabung ist mit 70% Ethanol sterilisiert Handschuhe. </li><li> Diese Lösung sollte klar sein. Es wird unter Argon-Gas, um Oxidation zu verhindern gespeichert. Wenn die Lösung trüb, dann mehrwandigen Bläschen erzeugt worden sind, und Sie müssen wieder von vorne anfangen. </li></ol> 2. Ermitteln, wie viel ICAM-1 und MHC auf der planaren Bilayer abgelagert werden <ol><li> Um das Experiment, ist es zunächst notwendig, um festzustellen, wie viel ICAM-1 und MHC auf einer gegebenen Fläche von Ni 2 + Chelat-Doppelschicht sind bei einer gegebenen Konzentration an löslichem Protein abgelagert. Um dies zu tun, Kaution Doppelschichten auf 5 Mikrometer Durchmesser Glasperlen, inkubieren Sie die Glasperlen mit Proteinlösungen unter Bedingungen, die annähernd denen in den Fluss-Zellen für die Bildgebung verwendet wird, und liest die Dichte des gebundenen Proteins durch eine Strömung immunofluorometric Assay. </li><li> FITC-markierten Antikörpern mit bekannten Zahlen von Fluorescein pro Molekül verwendet, um die Oberflächen-gebundenen Proteine ​​nachzuweisen. Fluorescein-Standard Kügelchen verwendet werden, um den Fluss microfluorimeter kalibrieren. </li><li> Wir schaffen Bedingungen, die annähernd denen von Antigen-präsentierenden Zellen mit 200 molecules/μm2 von ICAM-1 und 0,2-20 molecules/μm2 der I-Ek-MCC91-103-Komplex. </li></ol> 3. Reinigung der Glasdeckgläschen zur Bildung planarer Doppelschichten <ol><li> Planar Lipid-Doppelschichten bilden sich spontan, wenn die Liposomen Glaskolbensicherung, aber nur, wenn das Glas richtig gereinigt. </li><li> Bei der Arbeit mit Piranha, tragen wir die volle Schutzkleidung, einschließlich einer Säure Schürze und schwer, säurebeständigen Handschuhen. Sorgfältig trennen die Abfälle aus organischen Abfällen und ordnungsgemäß entsorgen. </li><li> Zur Vorbereitung der Säure Piranha-Lösung, 75 ml konzentrierte Schwefelsäure auf ein trockenes Becherglas und fügt vorsichtig 25 ml 30% iges Wasserstoffperoxid. Die Lösung schnell erwärmt sich auf mehr als 100 ° C. </li><li> Tauchen Sie den trockenen Deckgläser in die Lösung für 15 Minuten. Entfernen Sie aus der Lösung und spülen Sie in gereinigtem Wasser für eine Minute auf jeder Seite. Trocknen Sie die Deckgläser mit einem Vakuum-Quelle zur Ableitung des gereinigten Wassers. Lassen Sie das Piranha-Lösung zu kühlen, und fügen Sie den Piranha Abfallbehälter, die mit der Kappe lose, auch im Abzug markiert links ist. Wenn alle der Liposomen, Deckgläser und Proteine ​​hergestellt werden, ist der immunologischen Synapse bereit, gebildet werden. </li></ol> 4. Die Ausbildung der Doppelschichten <ol><li> Zur Ausbildung der Doppelschichten, nutzt unser Labor Bioptechs FCSII Kammern, die die Vorteile der integrierten Heizung haben. Die FCSII System basiert auf einem Sockel aus Edelstahl, die Bügelhälften einem mikrofluidischen Verteiler mit einer 0,5 mm Top-Dichtung, ein microaqueduct Rutsche mit Indium-Zinn-Oxid für die Heizung auf einer Oberfläche beschichtet, mit fluidischen Nut auf der anderen Seite, eine staubfreie 0,25 mm Dichtung, dass die Höhe der Flusskammer und der Piranha definiert gereinigt 40 mm runden Deckglas, auf denen die Doppelschicht gebildet wird. </li><li> Während das Deckglas, auf denen die Doppelschicht gebildet wird, muss stark hydrophil und sauber, die microacqueduct schieben tatsächlich funktioniert besser, wenn es mehr hydrophoben ist. Making the microaqueduct mehr hydrophobe durch Behandlung mit 1% HSA 60 Minuten bei Raumtemperatur erreicht wird, dann Waschen mit reinem Wasser und Trocknen vollständig nach dieser Konditionierung, sowie zwischen den Anwendungen. Die Beschichtung von denaturierten Proteinen durch dieses Verfahren verlassen können 1 ul Liposomensuspension zu einer Runde hemisphaerica Forml Tropfen,&gt; 0,25 mm hoch ist. </li><li> Um die Durchflusszelle und bilden planare Doppelschichten montieren, stellen die vielfältigen, mit den Rohren nach oben gerichtet, auf eine ebene Fläche. Legen Sie dann die 0. 5 mm Dichtung mit Löchern in den fluidischen Röhren positioniert, um sicherzustellen, dass es sitzt flach. Sanft legen Sie die microaqueduct Seite auf die Dichtung mit dem fluidischen Kanäle nach oben und stellen Sie sicher, es Sitze ohne jeglichen Druck nach unten flach, bis die Überprüfung, dass die Löcher in die Folie mit dem mikrofluidischen Rohre auszurichten. Legen Sie die staubfreie 0,25 mm Dichtung mit einem rechteckigen Ausschnitt auf der Oberseite des microaqueduct schieben, so dass die Kanal mit dem fluidischen Kanälen gesäumt und es gibt keine größeren Lufträumen. </li><li> Legen Sie bis zu 5 x 1 ul Tropfen Liposomensuspension auf der Oberfläche des microaqueduct Folie in ein Muster, so dass die Mitte-zu-Mitte-Abstand zwischen den einzelnen Tropfen 2,5 mm ist. Die 5 Liposomen Tropfen können unterschiedliche Zusammensetzungen haben, aber in diesem Fall werden wir nur bilden zwei Doppelschichten, eine ohne Ni 2 + + Komplexbildner Lipide und eine mit 10% Ni 2 + + Komplexbildner Lipiden. </li><li> Sobald diese Tropfen vorhanden sind, vorsichtig den Deckel nach unten rutschen auf der Oberfläche in einer Bewegung. Clamp auf den Boden so schnell wie möglich. Das Liposom Tropfen sollten Kontakt mit dem Deckglas zu machen. Dieser Kontakt sollte nicht gebrochen werden, da die Doppelschichten wahrscheinlich schon gebildet haben; etwaige Störungen können in defekten Doppelschichten führen. </li><li> Es ist wichtig, sich daran zu erinnern, um die Positionen der Doppelschichten mit ein paar kleinen Tinte Flecken auf der microacqueduct Folie Positionierung des Deckglas unter dem Mikroskop Hilfe zu markieren. Warten Sie 20 Minuten, um sicherzustellen, hat das System äquilibriert. Übertragen Sie alle Lösungen mit einer 20 ml Spritze verbunden, über 20 cm Schlauch und einem 3-Wege-Hahn. Schließen Sie einen anderen Port des Hahnes auf die Messzelle mit einem kurzen Rohrstück das Totvolumen zwischen dem Hahn und der Durchflusszelle zu minimieren. Füllen Sie die Spritze mit HEPES-gepufferte Kochsalzlösung mit 2 mM MgCl 2, 1 mM CaCl 2, 5 mM Glucose und 1% humanem Serumalbumin. </li><li> Prime den Schlauch und Hahn so gibt es keine Luftblasen. Schließen Sie hier die Messzellen und durchzusetzen 5 ml in einer Bewegung, während Sie sicherstellen, dass keine Luftblasen in den Doppelschicht passieren. Jetzt haben Sie Ihre Doppelschichten. </li></ol> Dieses Video Artikel unterstützt &quot;Hunter Gatherer und zurück: immunologischen Synapsen und Kinapses als Variationen über das Thema der amöboiden Locomotion&quot; von <a href="http://arjournals.annualreviews.org/action/doSearch?action=runSearch&amp;type=advanced&amp;result=true&amp;prevSearch=%2Bauthorsfield%3A%28Dustin%2C+Michael+L.%29" target="_blank">Michael L. Dustin</a> , <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.cellbio.24.110707.175226">Annual Review of Cell and Developmental Biology</a> , Band 24, 2008.

supported planar bilayers for the formation of study of immunological synapses and kinapse

Unterstützte planaren Doppelschichten sind leistungsfähige Werkzeuge, die verwendet werden, um die molekularen Wechselwirkungen in einer immunologischen Synapse Modell werden können. Zur Simulation der Wechselwirkungen zwischen Lymphozyten und Antigen-präsentierende Zellen, verwenden wir Ni2 +-Chelat-Lipide zur rekombinanten Zell-Adhäsion und MHC-Proteine ​​an der oberen Flugblatt einer Doppelschicht mit Poly-Histidin-tags zu verankern. Planar-Doppelschichten sind durch die Vorbereitung Lipid, die Behandlung der Glasflächen, wo der Doppelschicht bilden, und dann bildet der Doppelschicht in einer spezialisierten Kammer mit einem Flow-Zelle, wo die Lymphozyten hinzugefügt werden generiert. Dann Doppelschichten sind mit Ni-und His-markierten rekombinanten Proteinen berechnet werden hinzugefügt. Schließlich Lymphozyten sind in die Messzelle und TIRF-Mikroskopie injiziert werden, um Bild Synapsenbildung und die Mechanismen genutzt werden, dass die Kontrolle T-Zell-Lokomotion, Websites von Rezeptor-Sortierung und Websites von Rezeptor-Abbau.

Watch this Scientific Journal Video about Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse at JoVE.com

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Unterstützte planaren Doppelschichten sind leistungsfähige Werkzeuge, die verwendet werden, um die molekularen Wechselwirkungen in einer immunologischen Synapse Modell werden können. Hier zeigen wir Methoden zur Verankerung Zelladhäsionsproteine ​​bekannt zu modulieren Synapsenbildung an der oberen Flugblatt der Lipid bilyer und visualisieren Synapsenbildung mit TIRF-Mikroskopie.

Unterstützte Planar-Membranen für die Bildung von Study of immunologischen Synapsen und Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

supported-planar-bilayers-for-formation-study-immunological-synapses

Research

JoVE Journal

Biology

12.5K Aufrufe.  New York University - NYU. Unterstützte planaren Doppelschichten sind leistungsfähige Werkzeuge, die verwendet werden, um die molekularen Wechselwirkungen in einer immunologischen Synapse Modell werden können. Hier zeigen wir Methoden zur Verankerung Zelladhäsionsproteine ​​bekannt zu modulieren Synapsenbildung an der oberen Flugblatt der Lipid bilyer und visualisieren Synapsenbildung mit TIRF-Mikroskopie.

Serie: Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Forschung

Biologie

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated-Tiermodell Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Herstellung künstlicher Doppelschichten für Elektrophysiologie Experimente

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Methoden zur Untersuchung des Zebrafisch Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolierung und Reinigung von Drosophila Peripheren Neuronen durch Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Einrichtung zur Rückgewinnung von kryokonservierten Proben versehentlich fallengelassen in Kryo-Lagerbehälter

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Direct Imaging von Calcium ER mit Targeted-Esterase Induzierte Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Bäckerhefe Protein Extraktion und Reinigung für das Studium der Funktion, Wechselwirkungen, und post-translationale Modifikationen

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Endothelzellen Schlauchbildung Assay für die<em&gt; In-vitro-</em&gt; Studium der Angiogenese

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

Hochdurchsatz-Analyse von Bild Tumorsphäroide: Eine benutzerfreundliche Software-Anwendung, um die Größe der Sphäroide automatisch und präzise messen

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...