Wir danken den Mitgliedern des Matsunami Labor für die kritische Durchsicht des Manuskripts.

1. Vorgehensweise: Das Verfahren besteht aus drei Teilen über eine Gesamtzeit von 3 Tagen abgeschlossen: Übertragung von Zellen, Transfektion und Immunzytochemie, einen Schritt pro Tag durchgeführt. Transfer und Transfektion von Zellen in den ersten zwei Tagen muss unter sterilen Bedingungen in einer Laminar-Flow-Kammer durchgeführt werden. Tag 1: Transfer von HEK293T Zellen für Oberflächen-Expression Assays. <ol><li> Übertragungsmedium (M10): Mix Minimum Essential Medium mit 10% (nach Volumen) fötalem Rinderserum in sterilem Zustand ergänzt. </li><li> Wachsen Zellen, um eine gewünschte Konfluenz in einer 100mm Kulturschale, abhängig von der Anzahl der Platten für die Einrichtung. Es ist wichtig, den Anteil der Zellen zu übertragen überwucherte oder spärlich Zellen zu vermeiden bestimmen: zum Beispiel aus einer 100% konfluenten 100 mm Schale, kann man etwa 3% bis 35 mm Schale Transfer zu ca. 30% Konfluenz in den erhalten Letztere. </li><li> Vor der Übertragung Zellen, in den sterilen Laminar-Flow-Kammer, Mantel 22 X 22 mm Glasdeckgläschen mit steriler Poly D Lysin (1 mg / mL) und Platz eins in jedem 35 mm Zellkulturschale, beschichteten Seite nach oben für die Beschichtung Zellen (Abbildung 1 ). Lassen Sie die Lösung für 5 dry - 10 Minuten. Während dieser Zeit kann die UV-Quelle in der laminaren Kammer eingeschaltet werden, um die Deckgläser zu sterilisieren, wenn gewünscht. Poly D Lysin hilft heterologen Zellen Stick an die Deckgläser. </li><li> Zur Übertragung von Zellen, saugen aus den bestehenden Medien in der 100mm Schale, durch vorsichtige Zugabe von 8 ml sterilem PBS, PBS waschen und absaugen trypsinize die Zellen mit 3 ml 0,05% Trypsin-EDTA; wenn Zellen aus der Schale lösen, Block weiteren enzymatischen Reaktion durch Zugabe von 5 mL M10. Man reibt die Zellen und Transfer erforderlichen Volumens in einen sterilen konischen Rohr; Zentrifuge bei 200g für 5 Minuten die Zellen zu pelletieren. Absaugen aus Überstand mit Trypsin-EDTA, so dass die Pellets von Zellen intakt. Zur Übertragung der Zellen auf 35 mm-Schalen, 1 mL M10 für jedes Gericht und verreiben, um die Zellen zu distanzieren. 1 ml der resuspendierten Zellen zu jedem Gericht. Inkubieren Zellen über Nacht bei 37 ° C, 5% CO 2. <img src="/files/ftp_upload/2405/2405fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Coat 22 X 22 mm Deckgläser mit Poly-D Lysin und legen Sie eine beschichtete Seite nach oben in je 35 mm Schale. </li></ol> Tag 2: Transfektion von Zellen HEK293T. <ol><li> 18-24 Stunden nach der Übertragung werden die Zellen bereit für die Transfektion des Rezeptors an getestet werden. Verwenden Sie Rezeptor und RTP1S in Säugerexpressionsvektor PCI, aus Bakterienkulturen mit Qiagen Miniprep Kit nach Protokoll vor 4 beschrieben vorbereitet geklont. Verwenden Rezeptoren, N terminal mit 20 Aminosäuren langes Rhodopsin Epitop markiert, um Immunfärbung zu erleichtern. </li><li> Verwenden Sie 1000ng von Rezeptor-Konstrukt, 250 ng RTP1S konstruieren und 50 ng der grünen Fluoreszenz-Protein jeder Transfektion. Führen Sie die Transfektion mit Lipofectamine 2000, als pro-Hersteller Handbuch. </li></ol> Tag 3: Immunzytochemie auf der Zelloberfläche angefärbt Rezeptoren zu visualisieren. <ol><li> Vor Beginn der Vorbereitung Färbung und Waschlösungen und kühlen auf 4 ° C. Die Färbung Medium: Minimum Essential Medium, 45 ml; Fetal Bovine Serum, 5 ml; HEPES (1M), 500 ul (Endkonzentration 10 mM); Natriumazid (1,5 MB), 500 ul (Endkonzentration 15 mM). Waschlösung: HBSS, 500 mL; HEPES (1M), 5 ml (Endkonzentration 10 mM); Natriumazid (1,5 MB), 5 ml (Endkonzentration 15 mM). Beide Lösungen können bei 4 ° C zur Wiederverwendung gespeichert werden. Bereiten Antikörper werden durch Verdünnung benötigten Antikörper in der Färbung Medium 100-fach. </li><li> Starten Immunchemie für Oberflächen-Färbung für Geruchsrezeptoren etwa 24 Stunden nach der Transfektion. Um Lebendzellfärbungen, der experimentelle Aufbau muss auf 4 gekühlt werden durchführen ° C: Füllen Sie einen großen enthielt mit Eis und pat die Oberfläche des Eis, um es so gleichmäßig wie möglich zu machen, statt ein Tablett auf dem Eis und sprühen Sie es mit 70% Ethanol. Schneiden Sie ein Stück Parafilm (kleiner als die Länge des Fachs) und bringt es auf den Ethanol besprüht Tablett, um eine glatte Oberfläche zu erhalten. Übertragen Sie die Glasdeckgläschen mit transfizierten Zellen auf dem Parafilm, ein zu einer Zeit, Zelle nach oben, mit einer Pinzette. Layer-decken jeweils mit 100 ul kaltem primären Färbelösung slip, Extrudieren sorgfältig von einer Ecke des Schlupf (Abbildung 2). Nach Schichtung all die Deckgläser mit Antikörper-Lösung, decken Sie das Fach zu verhindern Austrocknen der Lösung in der Luft. Führen Sie primäre Inkubation für 45-60 Minuten. </li><li> Nach der Grundschule Inkubation schnell übertragen Deckgläser auf die ursprüngliche 35-mm-Kulturschalen, mit den Medien. Saugen Sie das Medien-und 2 mL gekühltes Waschlösung vorsichtig vom Rand des Tellers zu verhindern verlieren Zellen aus dem Deckglas. Absaugen der Waschlösung und wiederholen Sie zweimal auf drei Wäschen abzuschließen. Am Ende des dritten waschen, nicht saugen die Waschlösung aus. </li><li> Bereiten sekundären Antikörper Löauf in der Färbung Medien und übertragen Sie die Deckgläser aus der Waschlösung in eine neu angebrachte Parafilm Schicht in der gleichen Schublade. Wieder leicht extrudieren 100 ul Antikörper-Lösung auf Layer jedes Deckglas, aus der Ecke, wie die primären Inkubation. Führen Sie sekundäre Inkubation für 30 bis 45 Minuten und Transfer zurück zum Waschlösung in den ursprünglichen 35 mm Gerichte. Einmal mehr, waschen Sie die Deckgläser dreimal so beschrieben. </li><li> Saugen Sie Waschlösung aus dem letzten Wasch-und 2 mL 1% Paraformaldehyd gekühlt. Fix-Zellen in gekühltem 1% Paraformaldehyd-Lösung auf Eis für 15 Minuten. </li><li> Montieren Sie den Deckel rutscht auf saubere Mikroskop Objektträger mit Montage-Reagenz Mowiol, mit der Zelle nach unten und vorsichtig schließen alle Luftblasen (Abbildung 3). Sobald Mowiol Luft trocknet, waschen Deckgläser vorsichtig durch Zugabe von destilliertem Wasser auf der Oberfläche und Absaugen sofort. <img src="/files/ftp_upload/2405/2405fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Schicht jedes Deckglas mit 100 ul kaltem primären Färbelösung. <img src="/files/ftp_upload/2405/2405fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Montieren Sie die Deckgläser mit der Zelle nach unten auf saubere Objektträger aus Glas mit Mowiol Montage-Reagenz. </li></ol> 2. Repräsentative Ergebnisse: Lebendzellfärbungen ermöglicht einem, Proteine ​​auf der Oberfläche von Zellen sichtbar zu machen, auf die Transfektion von Rezeptoren mit Chaperone (in diesem Fall Geruchsrezeptor Olfr62 mit Rezeptor-Transport Protein RTP1S) (Abbildung 4). Zelloberflächenfärbung der Rezeptoren wird durch punktförmige Muster der Färbung (4B, Einschub) gekennzeichnet. Die Färbung durchgeführt unangemessen zu höheren Geräuschentwicklung führen, wodurch es schwierig für den Betrachter auf die reale Oberfläche Färbung von Rauschen zu unterscheiden. <img src="/files/ftp_upload/2405/2405fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Transfektion von Rezeptoren mit Chaperone ermöglicht die Visualisierung der Proteine ​​auf der Oberfläche von Zellen mit Hilfe von lebenden Zellen Färbung. Transfektion der Geruchsrezeptor Olfr62 allein (A) und die Transfektion von Olfr62 mit RTP1S Chaperon (B). GFP Expression dienen als Transfektion Kontrolle (A &#39;und B&#39;).

<ol>
	<li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65, 175-187 (1991).</li><li>Malnic, B., Hirono, J., Sato, T., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+receptor+codes+for+odors.">Combinatorial receptor codes for odors.</a> Cell. 96, 713-723 (1999).</li><li>Saito, H., Kubota, M., Roberts, R. W., Chi, Q., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTP+family+members+induce+functional+expression+of+mammalian+odorant+receptors.">RTP family members induce functional expression of mammalian odorant receptors.</a> Cell. 119, 679-691 (2004).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell-surface+expression+and+measuring+activation+of+mammalian+odorant+receptors+in+heterologous+cells.">Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.</a> Nat Protoc. 3, 1402-1413 (2008).</li></ol>

Jeder Schritt muss sorgfältig mit einem auffälligen und markanten Oberflächenbeschmutzung sicher durchgeführt werden. Die gesamte Färbung muss in kaltem durchgeführt werden (auf Eis), und das Tablett, auf dem Deckgläschen gelegt werden muss vor gekühlt werden, um zu verwenden, um sicherzustellen, Zellen am Leben bleiben. Außerdem Fixierung am Ende der Oberfläche Färbung durchgeführt. Dies steht in krassem Gegensatz zu internen Färbung, wo Fixierung Färbung an der Zellmembran zu primären und sekundären An...

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<td>Cover slip 22 X 22 mm (#1)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>72200-11</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
<td>&nbsp;</td>
<tr>
<td>Minimal essential medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M4655</td>
<td>With L glutamate, Earle&#x2019;s salt and bicarbonate</td>
</tr>
<tr>
<td>Mowiol</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>475904</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>EMS</td>
<td>19208</td>
<td>&nbsp;</td>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) 1X </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>14025</td>
<td>With calcium chloride and magnesium chloride</td>
</tr>
<tr>
<td>HEPES Buffer Solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Saline (PBS)</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-040-CV</td>
<td>Without calcium and magnesium</td>
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<tr>
<td>Poly D Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
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<td>Sodium Azide</td>
<td>&nbsp;</td>
<td>EM Science</td>
<td>SX0299-1</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dish 35 X 10 mm</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353801</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>0.05%</td>
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assaying surface expression of chemosensory receptors in heterologous cells

Die lebendige Welt der Gerüche ist durch den Sinn des Geruchssinns anerkannt. Der Geruchssinn Mäusen wird durch ein Repertoire von etwa 1200 G Protein-gekoppelte Rezeptoren (GPCRs) 1, die postuliert, dass flüchtige Duftmoleküle binden und Umwandlung der extrazellulären Signals in ein intrazelluläres Signal durch Kopplung mit G-Protein Gαolf vermittelt. Die Bindung des Geruchsstoffen an die Rezeptoren wird angenommen, dass eine kombinatorische Regel, dass man Geruchsstoff kann mehrere Rezeptoren binden und einen Rezeptor können verschiedene Geruchsstoffe in unterschiedlichem Maße 2 zu binden folgen. Biochemische, Signal-und Ligandenbindung Studien wurden bequemerweise für die meisten GPCRs unter Verwendung heterologen Zellen. Doch der heterologen Zellen verwenden für das Studium der Geruchsrezeptoren war für eine lange Zeit ausgeschlossen, da auf die Transfektionseffizienz sie an die Oberfläche zu exportieren ist fehlgeschlagen. Saito et al haben Einzel-Membran passieren Rezeptor Transport Protein (RTP) Familie Chaperone zeigen erhöhte Expression in den Riechzellen und fungieren als Chaperone für den Verkehr Geruchsrezeptoren auf der Oberfläche in heterologen Zellen, wenn co zusammen transfiziert 3 gezeigt. Zur Durchführung biochemischer Assays für Rezeptoren mit heterologen Zellen, muss man zunächst ermitteln, ob der Rezeptor zeigt robuste Oberfläche Ausdruck in der Zell-Linie. Dies kann durch die Überexpression der Rezeptoren mit dem Chaperon RTP1S durch Lebendzellfärbungen zu Fluoreszenz-Label der extrazellulären Domäne oder einen Tag in der extrazellulären Domäne ausschließlich gefolgt getestet werden. Hier zeigen wir ein Protokoll für die Durchführung Lebendzellfärbungen, die zur Geruchsrezeptoren auf der Oberfläche von Zellen HEK293T bequem nachgewiesen werden können. Darüber hinaus kann es auch zum Testen auf Oberflächenexpression von anderen Chemosensoren oder GPCRs verwendet werden.

Watch this Scientific Journal Video about Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells at JoVE.com

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Hier zeigen wir ein Protokoll für die Durchführung Lebendzellfärbungen, die zur Geruchsrezeptoren auf der Oberfläche von Zellen HEK293T bequem nachgewiesen werden können. Darüber hinaus kann es auch zum Testen auf Oberflächenexpression von anderen Chemosensoren oder GPCRs verwendet werden.

Assaying Oberfläche Expression von Chemosensoren in heterologen Zellen

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

assaying-surface-expression-chemosensory-receptors-heterologous

Research

JoVE Journal

Neuroscience

10.8K Aufrufe.  Duke University. Hier zeigen wir ein Protokoll für die Durchführung Lebendzellfärbungen, die zur Geruchsrezeptoren auf der Oberfläche von Zellen HEK293T bequem nachgewiesen werden können. Darüber hinaus kann es auch zum Testen auf Oberflächenexpression von anderen Chemosensoren oder GPCRs verwendet werden.

Serie: Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Assaying Bewegungsaktivität zu zirkadianen Rhythmen Study and Sleep Parameter in Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Forschung

Neurowissenschaften

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Assaying Bewegungsapparat, Lernen und Gedächtnis Defizite in Drosophila Modelle von Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Elektrophysiologie II: Extrazelluläre Saugelektrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-Cell-Imaging von Sinnesorgan Vorläuferzellen im Intact Drosophila Puppen

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Isolating Nasal Olfaktorische Stammzellen von Nagetieren oder Menschen

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Hochauflösende Live-Bildgebung von Zell-Verhalten in der Entwicklung von Neuroepithel

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Testen DNA Damage in Hippocampus-Neuronen mit dem Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Propriozeption und Spannungsrezeptoren in Crab Gliedmassen: Schülerlabor Übungen

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induktion von Plastizität Astrozytäre Rezeptoren durch Manipulation der neuronalen Feuerraten

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Bestimmung der Zelloberflächenexpression und der endozytischen Rate von Proteinen in primären Astrozytenkulturen unter Verwendung von Biotinylierung

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...