Name: organelle transport in cultured drosophila cells: s2 cell line and primary neurons.
Uploaded: 2013-11-20T18:00:00
Description: Watch this Scientific Journal Video about Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons. at JoVE.com

Vi takker medlemmerne af Gelfand lab, der har bidraget til udviklingen af ​​disse protokoller. Forskning rapporteret i denne publikation blev støttet af National Institute of General Medical Science af National Institutes of Health under tildelingen nummer R01GM052111 til VIG og baskiske regering Department of Education, universiteter og forskning under tildeling nummer BFI-2011-295 til UDC.

1. Drosophila S2-celler <ol><li> Fremstilling af ConA overtrukne dækglas <ol><li> Placer dækglas i en keramisk rack og vaske dem med chromsyre nedsænkning i 1 time. [ADVARSEL: chromsyre er ætsende og kan forårsage irritation af øjne, næse, hals og hud]. </li><li> Skyl dækglas grundigt med konstant kører dH2O i 30 min, indtil den sure er helt vasket ud. </li><li> Lad dækglas lufttørring og belægge dem med ConA (opløsning 0,5 mg / ml i dH2O) i 30 min. ...

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Her præsenterer vi detaljerede protokoller for at studere mikrotubuli-afhængig godstransport i Drosophila S2-celler og primære neuron kultur. Både S2 processer og neurites er strukturer fyldt med bundtede mikrotubuli arrays, der tjener som spor til organel transport. To strategier er typisk anvendt til at mærke organeller: transfektion af vektorer, der koder for et fluorescerende protein med en organel-targeting sekvens eller farvning med fluorescerende farvestoffer tilsat dire...

Drosophila S2-celler har vundet stigende popularitet til at studere mange cellulære processer. Disse celler blev oprindeligt stammer fra trypsinerede sene embryoner af OregonR Drosophila melanogaster og vedligeholde makrofaglignende funktioner 1.. Drosophila S2-celler har mange fordele frem for andre cellelinjer. De dyrkes ved 25 ° C uden CO 2, og kan afbildes i mange timer ved stuetemperatur uden behov for opvarmning eller gasudveksling. Endnu vigtigere er, Drosophila S2-celler er meget følsomme over for RNAi behandling giver høj effektivitet knockdown af proteiner af interesse. S2-celler er yderst transficerbare og kan vælges stabile cellelinier mindre end fire uger for at tillade stabil ektopisk ekspression af proteiner af interesse. S2-celler normalt vokse enten løst tilknyttet, men ikke spredes på en kolbe eller i suspension. De kan induceres til at spredes på dækglas belagt med en plante lectin concanavalin A (ConA). Periferien af ​​de spredte celler er specielt tynd og derfor er ideel til levende celler mikroskopi. Detaljerede protokoller for S2-celler kultur, RNAi behandling levende lys billeddannelse og immunfarvning kan findes i litteraturen 2,3. Vores laboratorium har vedtaget S2-celler som et modelsystem til at studere mikrotubulus-baserede godstransport. Drosophila-S2-celler behandlet med et lægemiddel, som depolymeriserer eller skærer F-actin, såsom cytochalasin D (CytoD), vokser lange processer fyldt med ensartet polariserede mikrotubuli 4 -10, hvilket gør det til et ideelt system til at studere last bevægelse langs mikrotubuli arrays. Forskellige parametre for transport i disse processer (. Dvs. fremskrivning længder, hastigheder, retningsbestemmelse osv.) kan let måles ved hjælp af automatiserede partikel-tracking software, DiaTrack (Semasopht: http://www.microscopy.info/Organization/Details/4423; Dr . Pascal Vallotton: http://www.csiro.au/en/Organisation-Structure/Divisions/Mathematics-Informatics-and-Statistics/PascalVallotton.aspx # a4). Disse egenskaber gør S2-celler en tiltalende system til at studere godstransport langs mikrotubuli i vævsdyrkningsceller. Pilotforsøg i S2-celler kan udvides til en fysiologisk vigtig model for transporterende studier i Drosophila primære neuroner. Svarende til S2-celler, neuroner dyrket fra dissocierede embryoner er permeabel for små molekyler, såsom farvestoffer og inhibitorer og høj opløsning til billeddannelse af organel transport kan udføres i neuroner ved stuetemperatur. Brug Drosophila med forskellige genetiske baggrunde, kan vi undersøge gen-funktion i ubearbejdet neuroner på knockout (genetiske tab af funktion mutationer), knockdown (dsRNA injektion eller udtryk) eller ektopisk udtryk (transgene fluer). Konkret betyder fragmentering af actin filamenter hjælp CytoD behandling ikke forhindre neuritudvækst, men i stedet de vokser hurtigere, og dermed kan vi studere mikrotubuli-dependent organel transport CytoD-behandlede neuroner uden indflydelse af actinfilamenter 11. Derfor primære neuronale kulturer kombinere fordelene ved vævsdyrkningsceller og flyve genetik, hvilket gør det en stor system til at studere godstransport i en fysiologisk relevant system, 10,12. Da flere familiær neurodegenerative sygdomme kan være forårsaget af eller associeret med transportenheder defekter (fx Parkinsons sygdom 13, Huntingtons sygdom 14. Alzheimers sygdom 15,16, Amyotrofisk Lateral Sclerose / ALS 17, HMN7B og Perry syndrom 18,19, og spinocerebellar ataksi typen 5/SCA5 20), kan primære neuronale kulturer være nyttige i analysen af virkningerne af specifikke mutationer forårsager disse sygdomme på mikrotubuli-afhængige transport. Endelig er der vigtige egenskaber ved mikrotubuli-afhængige transport velbevaret mellem pattedyr og fluer, men <em&gt; Drosophila genetisk og cellekultur er billigere og tidskrævende, der berettiger til anvendelse af dyrkede Drosophila celler til at studere vigtige aspekter af organel transport i normale og patologiske tilstande.

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			<td height="61" style="height:61px;width:124px;">Powder Insect cell medium (Schneider&#8217;s)</td>
			<td style="width:133px;">Sigma&#160;</td>
			<td style="width:179px;">S9895</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Insect &#8211;Xpress medium</td>
			<td style="width:133px;">Fisher</td>
			<td style="width:179px;">BW12730Q</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Insulin</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">I6634</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Penicillin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">P93000</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Streptomycin</td>
			<td style="width:133px;">Research Products International</td>
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			<td height="41" style="height:41px;width:124px;">Tetracycline hydrochloride</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">T7660-5G</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Fetal bovine serum</td>
			<td style="width:133px;">Atlanta Biologicals</td>
			<td style="width:179px;">S11150</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Concanavalin A</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">C2010</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Cytochalasin D</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">IC15077105</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:124px;">LysoTracker Red DND-99</td>
			<td style="width:133px;">Molecular Probes</td>
			<td style="width:179px;">L7528</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:124px;">100 &#181;m Cell strainer</td>
			<td style="width:133px;">Fisher Scientific</td>
			<td style="width:179px;">22363549</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:124px;">25 mm Circle cover glass</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">48380 080</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Drosophila Vials</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">89092-730</td>
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		<tr height="81">
			<td height="81" style="height:81px;width:124px;">Disposable pellet pestles with matching microtubes</td>
			<td style="width:133px;">Kimble Chase</td>
			<td style="width:179px;">749520-0000</td>
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organelle transport in cultured drosophila cells: s2 cell line and primary neurons.

Drosophila S2-celler belagt på et dækglas i tilstedeværelsen af actin-depolymerisering drug danne lange ugrenede processer fyldt med ensartet polariserede mikrotubuli. Organeller bevæge sig langs disse processer af mikrotubuli motorer. Nem vedligeholdelse, høj følsomhed til RNAi-medieret protein knock-down og effektiv procedure for at skabe stabile cellelinjer gøre Drosophila S2-celler en ideel model for at studere godstransport via direkte billedvisning. De opnåede resultater med S2-celler kan yderligere anvendes til en mere fysiologisk relevant system: axonal transport i ubearbejdet neuroner dyrket fra dissocierede Drosophila embryoner. Dyrkede neuroner vokse lange neuritter fyldt med bundtede mikrotubuli, meget ligner S2 processer. Ligesom i S2-celler, kan organeller i dyrkede neuroner visualiseres ved enten organel-specifik fluorescerende farvestoffer eller ved anvendelse af fluorescerende organel markører kodes af DNA injiceret i tidlige embryoner eller udtrykkes i transgenic fluer. Derfor kan organel transport let optages i neuroner dyrket på dækglas anvender levende billeddannelse. Her beskriver vi procedurer for dyrkning og visualisere godstransport i Drosophila S2-celler og primære neuroner. Vi mener, at disse protokoller gør begge systemer tilgængelige for laboratorier studerer godstransport.

Drosophila S2-celler, der er knyttet på Cona-belagt dækglas spredes ind i en flad rund &quot;pandekage&quot; form (figur 1A og 1C). Men når S2-celler udsået på dækglas i overværelse af CytoD og lov til at sprede for 2-3 timer, danner de lange tynde processer fyldt med parallelle mikrotubuli (figur 1B og 1D). Disse mikrotubuli har ensartet polaritet med plus-ender ud 7. Time-lapse fluorescens billeddannelse af Drosophila</e...

Watch this Scientific Journal Video about Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons. at JoVE.com

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2-celler og dyrkede neuroner er gode systemer til billeddannelse af motordreven organel transport in vivo. Her beskriver vi detaljerede protokoller til dyrkning både celletyper, deres billedbehandling og analyse af transport.

Organel Transport i dyrkede<em&gt; Drosophila</em&gt; Celler: S2 Cell Line og Primary neuroner.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

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Biology

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Preparation of Drosophila S2 cells for Light Microscopy

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Primary Culture of Rat Hepatic Cells

Isolering og Primær kultur Rotte levercellerne

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Levende Imaging af Apoptotisk Cell Clearance under<em&gt; Drosophila</em&gt; Embryogenese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Generering af en &quot;humaniseret&quot;<em&gt; Drosophila</em&gt; S2 cellelinie Følsom over for Farmakologisk Inhibering af kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Et grafisk brugergrænseflade til softwareassisteret sporing af proteinkoncentration i dynamiske cellulære protrusioner

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Måling af glukoseoptagelse og reaktion på insulinstimulering i<em&gt; In Vitro</em&gt; Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...