Name: imaging cell membrane injury and subcellular processes involved in repair
Uploaded: 2014-03-24T20:45:00
Description: Watch this Scientific Journal Video about Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair at JoVE.com

Dette arbejde blev støttet af National Institutes of Health Tilskud AR055686 og AR060836 og postdoc-stipendium til at e.Kr. af fransk Muskelsvindfonden Association (AFM). Den cellulære imaging facilitet udnyttes i denne undersøgelse er støttet af National Institutes of Health Tilskud HD040677.

1.. Imaging Cell Membrane Repair Brug Bulk (Glas perle) Anskydning Protokollen giver særskilt mærkning de sårede celler og dem, der ikke heler. Sætte tal på disse populationer af celler kræver brug af tre betingelser: 1.. Test (C1) - Celler får lov til at reparere i nærvær af Ca2 +, 2. Kontrol 1 (ingen skade C2) - Cellerne inkuberes i nærvær af Ca 2 +, men ikke såret, og 3. Kontrol 2 (ingen reparation C3) - celler får lov til at reparere i fravær af Ca ...

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Cellemembranen skade in vivo opstår på grund af en række fysiologiske stressfaktorer og flere eksperimentelle metoder er blevet udviklet til at efterligne disse. Heriblandt skade cellemembranen af adhærente celler ved at skrabe dem af skålen, eller ved passage gennem en smal boring sprøjte 9,10. Efter sådanne skader cellerne helbrede i suspension og ikke levet op til den ekstracellulære matrix, som de normalt gør i vævet. Atter andre, såsom brug af poredannende toksiner kemisk ændre celle...

Cellemembranen fastholder integritet af celler ved at tilvejebringe en barriere mellem cellen og det ekstracellulære miljø. En kemisk, elektrisk eller mekanisk stimulus, der overstiger den normale fysiologiske tærskel samt tilstedeværelsen af ​​invaderende patogener kan hver resultat i skade på cellemembranen og udløse en efterfølgende cellulært respons til at reparere denne skade. For at overleve disse skader til cellemembranen celler besidder en effektiv mekanisme til reparation. Denne mekanisme er calcium afhængig og involverer intracellulær handel med proteiner såsom annexiner og MG53 blandt andre samt subcellulære rum såsom endosomer, lysosomer, Golgi afledte vesikler og mitokondrier til sårede cellemembranen 1-7. Dog forbliver detaljerne i rækkefølgen af ​​molekylære og subcellulære begivenheder involveret i reparation af beskadigede cellemembranen dårligt forstået. Celle reparation svar kan inddeles i øretly og sene svar. Tidlige reaktioner, der opstår inden for sekunder til minutter tidsskala, er uhyre vigtige i at bestemme arten af ​​sene reaktioner, der fører til en vellykket celle reparation eller celledød. End point assays baseret på bulk biokemiske og cellulære analyser har hjulpet med at etablere inddragelse af molekylære og cellulære processer i reparation. Men på grund af den heterogenitet og hurtigheden af ​​cellulære reparation respons, slutpunkt assays undlader at give de kinetiske og rumlige oplysninger om rækkefølgen af ​​begivenheder, der fører til at reparere. Tilgange, der muliggør kontrolleret skade cellemembranen og tillader overvågning af cellemembranen reparation og tilhørende subcellulære reaktioner ved høj rumlig og tidsmæssig opløsning er velegnet til sådanne undersøgelser. Her har sådanne tiltag blevet præsenteret. To af de protokoller, beskriver metoder til at overvåge den reelle tid kinetik cellemembran reparation og subcellulære reaktioner forbundet med reparationen i levende celler efter laser injUry. Som et supplement til disse levende celler baserede assays har slutpunkt assays også blevet beskrevet, der giver en population baseret foranstaltning til overvågning reparation af de enkelte celler og de tilknyttede subcellulære svar. For at demonstrere deres anvendelighed disse tilgange er blevet anvendt til at overvåge menneskehandel og exocytose af lysosomer som reaktion på cellemembranen skade.

<table border="0" cellpadding="0" cellspacing="0" width="814" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H1387</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP410</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>FITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1820</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>EMS</td>
			<td>15710</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>1/10 000 in PBS</td>
		</tr>
		<tr>
			<td>FM dye</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td>1 &#181;g/&#181;l in CIM or PBS</td>
		</tr>
		<tr>
			<td>FITC dextran</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>2 mg/ml in growth medium</td>
		</tr>
		<tr>
			<td>LAMP1&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-19992</td>
			<td>1/300 in growth medium</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 chicken anti-rat IgG</td>
			<td>Invitrogen</td>
			<td>A21470</td>
			<td>1/500 in blocking solution</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-545-86</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom petridish</td>
			<td>MatTek corporation</td>
			<td>P35G-1.0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone O ring</td>
			<td>Bellco</td>
			<td>1943-33315</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip holder</td>
			<td>Bellco</td>
			<td>1943-11111</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen</td>
			<td>A-7816</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>12001-566</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>VWR</td>
			<td>MP1400-500H</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Penicillin/Sreptomycin</td>
			<td>VWR</td>
			<td>12001-350</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>5% chicken serum in CIM is used for blocking solution during immunostaining</td>
		</tr>
		<tr>
			<td>PBS (Ca2+ and Mg2+ free)</td>
			<td>VWR</td>
			<td>12001-664</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Olympus America, PA</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>TIRF illuminator&#160;</td>
			<td>Olympus America, PA</td>
			<td>Cell^TIRF (IEC60825-1:2007)</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence illuminator</td>
			<td>Sutter Instruments, Novato CA</td>
			<td>Lambda DG-4 (DG-4 30)</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Photometrics, Tucson, AZ</td>
			<td>Evolve 512 EMCCD&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition and anaysis software</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Slidebook 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulsed laser</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Ablate (3iL13)</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokaihit, Japan</td>
			<td>INUBG2ASFP-ZILCS</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cell membrane injury and subcellular processes involved in repair

Evnen af ​​beskadigede celler til at helbrede er en grundlæggende cellulære proces, men cellulære og molekylære mekanismer, der er involveret i heling såret celler er dårligt forstået. Her assays er beskrevet til at overvåge evne og kinetik af heling af dyrkede celler efter lokaliseret skade. Den første protokol beskriver et slutpunkt tilgang til samtidig at bedømme cellemembranstatus reparation evne hundredvis af celler. Den anden protokol beskriver en real time scanning tilgang til at overvåge kinetik cellemembranen reparation i de enkelte celler efter lokaliseret skade med en pulserende laser. Som healing såret celler involverer handel med specifikke proteiner og subcellulære rum til stedet for skade, den tredje protokol beskriver brugen af ​​ovennævnte slutpunkt tilgang til at vurdere en hændelse menneskehandel (lysosomale exocytose) i hundredvis af celler sårede samtidigt og den sidste protokol beskriver anvendelsen af ​​pulserende laser skade sammen med TIRF mikroerkopi til at overvåge dynamikken i de enkelte subcellulære rum i skadede celler ved høj rumlig og tidsmæssig opløsning. Mens de protokoller her beskriver anvendelsen af ​​disse metoder til at undersøge sammenhængen mellem cellemembranen reparation og lysosomal exocytose i dyrkede muskelceller, kan de anvendes som sådanne til andre adhærerende dyrkede celle og subcellulært afsnit af valg.

Protokoller, der er beskrevet her for enkelt cell imaging er at overvåge evne og kinetik af cellemembranen reparation (protokol 1 og 2), og den subcellulære handel og fusion af lysosomer under reparation (Protokollerne 3 og 4). Protokol 1 viser en bulk assay, der tillader mærkning af alle tilskadekomne celler og identificere de tilskadekomne celler, der har undladt at reparere. Resultaterne i figur 1 viser, at medens de ubeskadigede celler (figur 1A) forb...

Watch this Scientific Journal Video about Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair at JoVE.com

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Processen med healing sårede celler involverer handel med specifikke proteiner og subcellulære rum til stedet for cellemembranen skade. Denne protokol beskriver assays til at overvåge disse processer.

Imaging Cell Membrane Skade og Subcellulære processer involveret i reparation

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

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