1. Preparare i lipidi <ol><li> Al fine di collegare le proteine ​​il foglio superiore di un doppio strato, usiamo Ni 2 + lipidi chelanti, che ancorano proteine ​​ricombinanti con poli-istidina tag. Alcuni tipi di cellule possono aderire in modo non specifico per Ni 2 + rivestite doppi strati, ma le cellule T generalmente non, rendendo questo metodo ottimale per studiare l&#39;attivazione delle cellule T. </li><li> La soluzione doppio strato lipidico è costituito da preparando una soluzione di 20% di Ni 2 + lipidi chelante fosfatidilcolina e 80% per l&#39;importo adeguato a un tubo di vetro con 1-2 ml di cloroformio-metanolo. <ol><li> Far evaporare il solvente sotto un flusso di azoto in un ambiente caldo (30-37 ° C), acqua sporca. Questo richiede di solito circa 15 minuti. </li><li> Rimuovere eventuali solvente rimanente sotto alto vuoto, utilizzando un liofilizzatore per 90 minuti. </li></ol></li><li> Sciogliere i lipidi nel 2% diottile-glucoside detergente in tampone Tris-salino, che crea una soluzione di detergente misto / fosfolipidi micelle. La concentrazione dei fosfolipidi finale dovrebbe essere 0,4 mm. Per formare liposomi, Dializzare di questa soluzione con 3 cambi di Tris-salino per rimuovere liposomi detergente e forma. Di solito lavoro con volumi dell&#39;ordine di 1 ml con 6 mm di diametro Spectra / Por # 2 tubi con un cut-off di circa 10 kDa. Stiamo attenti a escludere qualsiasi s bolle d&#39;aria per il fissaggio del tubo. Noi di solito sterile-filtro questa soluzione e fare la dialisi in condizioni di pulizia, la movimentazione usando guanti di etanolo al 70% sterilizzati. </li><li> Questa soluzione dovrebbe essere cristallino. E &#39;conservato in gas argon per evitare l&#39;ossidazione. Se la soluzione è torbida, poi multi-parete vescicole sono stati generati, e sarà necessario ricominciare da capo. </li></ol> 2. Determinare la quantità di ICAM-1 e MHC si depositano sul doppio strato planare <ol><li> Per impostare l&#39;esperimento, è prima necessario determinare la quantità di ICAM-1 e MHC sono depositate su una superficie dato di Ni 2 + chelanti bilayer a una data concentrazione di proteina solubile. Per fare questo, bilayers deposito il 5 micrometri di diametro perle di vetro, incubare le perle di vetro con le soluzioni di proteine ​​che approssimano quelli delle celle di flusso utilizzato per l&#39;imaging, e leggere la densità delle proteine ​​da un saggio di flusso immunofluorometric. Condizioni </li><li> Gli anticorpi coniugati con fluoresceina con un numero noto di fluorescina per molecola vengono utilizzati per rilevare la superficie è legato proteine. Fluoresceina sfere standard sono utilizzati per calibrare il microfluorimeter flusso. </li><li> Creeremo le condizioni che approssimano quelle cellule presentanti l&#39;antigene con 200 molecules/μm2 di ICAM-1 e 0,2-20 molecules/μm2 di I-Ek-MCC91-103 complessi. </li></ol> 3. Pulizia del vetro di copertura scivola per la formazione di doppi strati planari <ol><li> Doppi strati planari forma spontaneamente quando i liposomi fusibile di vetro, ma solo se il bicchiere sia ben pulito. </li><li> Quando si lavora con Piranha, abbiamo indossare idonei indumenti protettivi, tra cui un grembiule acido e pesante, guanti resistenti agli acidi. Con attenzione separare i rifiuti da rifiuti organici e smaltire in modo corretto. </li><li> Per preparare la soluzione Piranha acido, aggiungere 75 ml di acido solforico concentrato in un recipiente asciutto, ed aggiungere con cautela 25 ml di acqua ossigenata al 30%. La soluzione si riscalda rapidamente fino a oltre 100 ° C. </li><li> Immergere i coprioggetti secco nella soluzione per 15 minuti. Rimuovere dalla soluzione e sciacquare con acqua purificata per un minuto su ogni lato. Asciugare il coprioggetto utilizzando una fonte di vuoto per drenare l&#39;acqua purificata. Lasciare che la soluzione Piranha raffreddare e aggiungere al contenitore dei rifiuti Piranha, che è lasciata con il tappo sciolti, ben marcato nella cappa. Quando tutti i liposomi, coprioggetto, e le proteine ​​sono preparati, la sinapsi immunologica è pronto per essere formata. </li></ol> 4. Formare i doppi strati <ol><li> Per formare i doppi strati, il nostro laboratorio utilizza Bioptechs FCSII camere, che hanno il vantaggio di riscaldamento integrato. Il sistema FCSII è su una base in acciaio inox fascette insieme un collettore microfluidica con una guarnizione 0,5 millimetri superiore, una diapositiva microaqueduct rivestito con ossido di indio e stagno per il riscaldamento su una superficie, con scanalatura fluidico dall&#39;altro lato, una polvere 0,25 millimetri guarnizione che definisce l&#39;altezza della camera di flusso e il Piranha pulito coprioggetto 40 mm rotondo sul quale si forma il doppio strato. </li><li> Mentre il vetrino, sul quale si forma il doppio strato, deve essere altamente idrofili e pulito, la diapositiva microacqueduct effettivamente funziona meglio se è più idrofobo. Rendere il idrofobo microaqueduct più si ottiene trattando con l&#39;1% HSA per 60 minuti a temperatura ambiente, poi lavaggio con acqua pura e il completo indurimento dopo questo processo di condizionamento, così come tra usi. Il rivestimento di proteine ​​denaturate lasciata da questa procedura consente di 1 ml di sospensione di liposomi per formare una rotonda, hemispherical goccia che è&gt; 0,25 mm di altezza. </li><li> Per assemblare la cella a flusso e la forma doppi strati planari, posto il collettore, con i tubi diretta verso l&#39;alto, su una superficie piana. Poi, collocare il 0. Guarnizione 5 mm con fori posizionato sopra i tubi fluidico, assicurandosi che sia seduto piatto. Posizionare con delicatezza il lato microaqueduct sulla guarnizione con i canali fluidico rivolto verso l&#39;alto, e assicurarsi che i sedili piatto senza applicare alcuna pressione verso il basso, fino a verificare che i fori nella diapositiva allinearsi con i tubi microfluidica. Stendere la polvere guarnizione 0,25 mm con un taglio rettangolare sulla parte superiore della diapositiva microaqueduct modo che il canale sia in linea con i canali fluidici e non ci sono spazi d&#39;aria principali. </li><li> Posto fino a 5 gocce x 1 ml di sospensione liposomi sulla superficie del vetrino microaqueduct in un modello tale che il centro a centro distanza tra ogni goccia è di 2,5 mm. Il 5 gocce di liposomi possono avere composizioni diverse, ma in questo caso ci sarà solo formano due doppi strati, uno senza Ni 2 + + lipidi chelante e uno con il 10% Ni 2 + + lipidi chelanti. </li><li> Una volta che queste gocce sono in atto, posizionare con attenzione il coperchio scivolare sulla superficie in un unico movimento. Morsetto nella base il più rapidamente possibile. Le gocce liposomi dovrebbe entrare in contatto con la polizza di copertura. Questo contatto non deve essere spezzato, dal momento che bilayers hanno probabilmente già formate, qualsiasi interruzione può provocare doppi strati difettoso. </li><li> E &#39;importante ricordarsi di segnare le posizioni dei doppi strati con pochi punti di inchiostro piccolo della diapositiva microacqueduct ausilio per il posizionamento del vetrino sul microscopio. Attendere 20 minuti per assicurarsi che il sistema è equilibrato. Trasferire tutte le soluzioni utilizzando una siringa da 20 ml collegata a circa 20 cm di tubo flessibile ed un rubinetto a 3 vie. Collegare un altro porto del rubinetto di arresto nella cella di flusso per un breve tratto di tubo ridurre al minimo il volume morto tra il rubinetto e la cella di flusso. Riempire la siringa con HEPES soluzione tampone contenente 2 mM MgCl 2, 1 mM CaCl 2, 5 mM di glucosio e 1% di albumina umana. </li><li> Primo il tubo e rubinetto quindi non ci sono bolle d&#39;aria. Collegare questo alle cellule di flusso e pressione a 5 ml in un unico movimento mentre si guarda per assicurarsi che bolle d&#39;aria passare il doppio strato. Ora, avrete il vostro doppi strati. </li></ol> In questo articolo video supporta &quot;Hunter Gatherer e Back: Le sinapsi immunologica e Kinapses come variazioni sul tema di locomozione ameboide&quot; di <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 di Biologia Cellulare e dello Sviluppo</a> , Volume 24, 2008.

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

Supportati doppi strati planari sono strumenti potenti che possono essere utilizzati per modellare le interazioni molecolari in una sinapsi immunologica. Per simulare le interazioni tra i linfociti e le cellule presentanti l&#39;antigene, si usa Ni2 +-chelante lipidi per ancorare l&#39;adesione delle cellule e proteine ​​ricombinanti MHC il foglio superiore di un doppio strato di poli-istidina tag. Doppi strati planari sono generate da preparazione dei lipidi, trattando le superfici di vetro, dove si forma il doppio strato, e poi formando il doppio strato in una sezione specializzata che contiene un flusso di celle dove i linfociti saranno aggiunti. Poi, doppi strati sono accusati di Ni e il suo-tagged proteine ​​ricombinanti sono aggiunti. Infine, i linfociti vengono iniettati nella cellula di flusso e microscopia TIRF possono essere utilizzati per la formazione di sinapsi immagine ed i meccanismi che controllano la locomozione delle cellule T, siti di smistamento dei recettori, e siti di degradazione del recettore.

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

Supportati doppi strati planari sono strumenti potenti che possono essere utilizzati per modellare le interazioni molecolari in una sinapsi immunologica. Qui vi mostriamo i metodi per l&#39;ancoraggio delle proteine ​​di adesione cellulare conosciuto per modulare la formazione di sinapsi il foglio superiore della bilyer lipidi e visualizzare formazione di sinapsi usando la microscopia TIRF.

Supportato bilayers Planar per la formazione degli Studi di sinapsi immunologica e 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

Nanyang Technological University

Research

JoVE Journal

Biology

12.5K Views.  New York University - NYU. Supportati doppi strati planari sono strumenti potenti che possono essere utilizzati per modellare le interazioni molecolari in una sinapsi immunologica. Qui vi mostriamo i metodi per l'ancoraggio delle proteine ​​di adesione cellulare conosciuto per modulare la formazione di sinapsi il foglio superiore della bilyer lipidi e visualizzare formazione di sinapsi usando la microscopia TIRF.

Video: 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.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

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

Ricerca

Biologia

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 modello animale Stimoli

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.

Preparazione di doppi strati artificiali per esperimenti Elettrofisiologia

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.

Metodi per lo studio del mascellare Zebrafish 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.

Isolamento e purificazione di Drosophila Neuroni periferici da Magnetic Ordinamento Bead

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 dispositivo per il recupero di campioni crioconservati cadere accidentalmente nelle cisterne criogeniche

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.

Imaging diretta di calcio ER con Targeted-esterasi Induced Loading Dye (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.

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

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.

Endoteliali Tubo cellulare saggio Formazione per la<em&gt; In Vitro</em&gt; Studio di Angiogenesi

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.

High-throughput Immagine Analisi di sferoidi tumorali: Un software applicativo di facile utilizzo per misurare le dimensioni del Spheroids automaticamente e con precisione

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