Name: generating primary fibroblast cultures from mouse ear and tail tissues
Uploaded: 2016-01-10T13:00:00
Description: Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

Musene blev opstaldet i patogenfrie betingelser i overensstemmelse med de institutionelle retningslinier indtil euthanization (Institutional Animal Care og brug Udvalg (IACUC) retningslinjer på National University of Singapore og det nationale rådgivende udvalg for Laboratory Animal Research (NACLAR) retningslinjer). 1. Mus <ol><li> Bestil en mus af den passende genetisk baggrund. Denne protokol er baseret på væv afledt fra en C57BL / 6 mus. </li></ol> 2. Fremstilling af komplet mediu...

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Her giver vi en enkel, billig og hurtig eksperimentelle procedure for at etablere primære fibroblastkulturer fra ører og haler af mus. Ekstraktionen bør resultere i adhærente og hurtigt delende fibroblaster inden for 3 dage efter isolering af vævet. En vigtig begrænsning af primærelementer er degeneration, en permanent vækst anholdelse 15. Under anvendelse af protokollen, kan fibroblastkulturer passeres i 5 til 6 gange før fibroblaster bliver aldrende, angivet ved udfladning af celler, vokse i størr...

Primære celler er afledt af levende væv og dyrkes under in vitro betingelser. Det er almindeligt antaget, at primære celler mere ligner fysiologiske tilstand og den genetiske baggrund af det væv, hvorfra de stammer end udødeliggjorte eller tumorcellelinier 1. Af denne grund udgør primære celler en nyttig model til undersøgelse af biologiske spørgsmål 2,3. Men i modsætning til etablerede cellelinjer, der vokser i det uendelige, primære celler til sidst undergår ældning i kultur og skal ofte genetableres. Almindeligt anvendte primære celler indbefatter fibroblaster, epitelceller, endotelceller, T-celler, B-celler, knoglemarv-afledte makrofager (BMDM) og knoglemarv-afledte dendritiske celler (BMDC). Fibroblaster er ofte anvendes som primær cellekultur model. De tilbyder vigtige fordele frem for andre primære celler. Cellekulturer let etableres, let vedligeholdes og kræver ingen rensningsmedietion af celler før dyrkning. De har hurtig indledende spredning og ingen krav om specialiserede medium eller aktivering protokoller. Fibroblaster kan effektivt transficeres ved hjælp af biologiske, kemiske og fysiske protokoller 4,5. Der er en mulighed for at lagre ører i op til 10 dage ved stuetemperatur forud for etableringen af ​​cellekulturer. Fibroblastkulturer er fremmende for visualisering af cytoplasmatiske og egnet til omprogrammering ind induceret pluripotente stamceller (IPS) celler 6. Fibroblaster er vigtige celler i bindevæv, der udskiller kollagen proteiner og ekstracellulære matrix 7. De giver de strukturelle rammer i mange væv 8 og spille en væsentlig rolle i sårheling og væv reparation 9,10. Her beskriver vi en enkel og hurtig (&lt;4 timer) protokol til at etablere fibroblastkulturer fra ører og haler af mus 11. Protokollen kræver minimal musoplevelse at høste væv (i modsætning til andre protokoller 12,13) ​​og kan bruges til at etablere kulturer fra ørerne lagret i medium ved stuetemperatur i op til 10 dage.

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI-1640</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SH30027.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Calf Serum</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Asparagine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">107017</td>
			<td>Absolute for analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:140px;">Roche Diagnostics</td>
			<td style="width:119px;">11088866001</td>
			<td>From Clostridium histolyticum, lyophilized, non-sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pronase protease</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">53702</td>
			<td>From Streptomyces griseus&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris buffer (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">1415</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5M EDTA (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-1053</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10X Phosphate Buffered Saline (PBS)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-2040-10X4L</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA solution 10X</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">59418C-100ML</td>
			<td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amphotericin B</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2492-20ml</td>
			<td>250 &#956;g/ml in deionized water, sterile-filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forcep</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;">AE-BD312R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#956;M syringe filter</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 &#956;M cell strainer</td>
			<td style="width:140px;">SPL</td>
			<td style="width:119px;">93070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe plunger</td>
			<td style="width:140px;">Terumo</td>
			<td style="width:119px;">SS+10L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryovial tube</td>
			<td style="width:140px;">NUNC</td>
			<td style="width:119px;">368362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.7 ml microcentrifuge tube</td>
			<td style="width:140px;">Axygen</td>
			<td style="width:119px;">MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 cm cell culture dish</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">664160</td>
			<td>Cell culture treated dish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:140px;">GFL</td>
			<td style="width:119px;">1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:140px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubation shaker</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;"></td>
			<td>Certomat-BS1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axiovert 25 light microscope</td>
			<td style="width:140px;">Carl Zeiss AG</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating primary fibroblast cultures from mouse ear and tail tissues

Primære celler er afledt direkte fra væv og menes at være mere repræsentativ for den fysiologiske tilstand af celler in vivo end etablerede cellelinjer. Men primære cellekulturer har normalt en begrænset levetid og skal ofte genetableres. Fibroblaster er en let tilgængelig kilde til primære celler. Her diskuterer vi en enkel og hurtig eksperimentelle procedure for at etablere primære fibroblastkulturer fra ører og haler af mus. Protokollen kan bruges til at etablere primære fibroblastkulturer fra ører opbevaret ved stuetemperatur i op til 10 dage. Når protokollen er omhyggeligt fulgt, er usandsynlig på trods af anvendelsen af ​​ikke-sterilt væv opbevares i længere tid i nogle tilfælde forureninger. Fibroblaster formere sig hurtigt i kultur og kan udvides til et betydeligt antal før de undergik replikativ senescens.

Ekstraktion af fibroblaster fra væv resulterer i en betydelig mængde vævsrester (figur 1). I modsætning til væv vragrester, fibroblaster overholde væv kultur plastoverflader mellem dag 1 og 3 i kultur. Mediet af fibroblastkulturer kan sikkert ændret på dag 3 af kultur, der formodes at nedsætte mængderne af affald til stede i kulturen (figur 2). Fibroblaster vise en aflang morfologi og et klart synligt cytoplasma (figur 1 og 2). Mitotiske celle...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Vi beskriver en enkel og hurtig eksperimentelle procedure til generering af primære fibroblaster fra ørerne og haler af mus. Proceduren kræver ikke særlig dyretræningssystem og kan anvendes til generering af fibroblastkulturer fra ører opbevaret ved stuetemperatur i op til 10 dage.

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