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.

Mus ble plassert i patogenfrie vilkår i samsvar med de institusjonelle retningslinjer inntil euthanization (The Institutional Animal Care og bruk Committee (IACUC) retningslinjer ved National University of Singapore og National Advisory Committee for Forsøksdyrforskning (NACLAR) retningslinjer). 1. Mus <ol><li> Bestill ett muse av den aktuelle genetiske bakgrunn. Denne protokollen er basert på vev avledet fra en C57BL / 6 mus. </li></ol> 2. Utarbeidelse av komplett medium <ol><li> ...

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Her har vi tilveiebringe en enkel, billig og hurtig Fremgangsmåten for å etablere primære fibroblastkulturer fra ører og hale i mus. Ekstraksjonen bør resultere i adherente og raskt delende fibroblaster i løpet av 3 dager etter isolering av vevet. En viktig begrensning av primære celler er senescence, en permanent vekst arrest 15. Ved hjelp av protokollen, kan fibroblast kulturer skal passeres i 5 til 6 ganger før fibroblaster bli senescent, indikert av utflating av cellene, øker i størrelse (2-3 ga...

Primære celler som er avledet fra levende vev og dyrket under in vitro betingelser. Det er generelt antatt at primærceller ligner nærmere den fysiologiske tilstand og genetisk bakgrunn av vev hvorfra de stammer enn immortaliserte cellelinjer eller tumor 1. Av den grunn primære celler representerer en nyttig modell for å studere biologiske spørsmål 2,3. Men i motsetning til etablerte cellelinjer som vokser i det uendelige, primære celler til slutt gjennomgår alderdom i kultur og må ofte reetablert. Vanlig anvendte primære celler omfatter fibroblaster, epitelceller, endotelceller, T-celler, B-celler, benmarg-avledede makrofager (BMDM) og benmarg-avledede dendrittceller (BMDC). Fibroblaster blir ofte brukt som primærcellekultur modell. De tilbyr viktige fordeler fremfor andre primære celler. Cellekulturer kan lett etableres, opprettholdes lett og krever ingen purificasjon av celler før kultur. De har rask innledende spredning og ingen krav til spesialiserte middels eller aktiverings protokoller. Fibroblaster kan effektivt transfektert bruker biologiske, kjemiske og fysiske protokoller 4,5. Det er en mulighet for å lagre ører for opp til 10 dager ved RT forut for etablering av cellekulturer. Fibroblast kulturer er bidrar til visualisering av cytoplasmatiske prosesser og egnet for omprogrammering til induserte pluripotente stamceller (iPS-celler) 6. Fibroblaster er viktige celler i bindevevet som kollagen utskiller proteiner og ekstracellulær matriks 7. De gir den strukturelle rammer i mange vev 8 og spiller en viktig rolle i sårheling og vev reparasjon 9,10. Her beskriver vi en enkel og rask (4-t &lt;) protokoll for å etablere fibroblastkulturer fra ører og hale i mus 11. Protokollen krever minimal muserfaring for å høste de vev (i motsetning til andre protokoller 12,13) ​​og kan brukes til å etablere kulturer fra ørene som er lagret i mediet ved romtemperatur i opp til 10 dager.

<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 som er avledet direkte fra vev, og er antatt å være mer representativ for den fysiologiske tilstanden til cellene in vivo enn etablerte cellelinjer. Imidlertid primære cellekulturer som regel har en begrenset levetid og må ofte reetablert. Fibroblaster er en lett tilgjengelig kilde til primære celler. Her har vi diskuterer en enkel og rask Fremgangsmåten for å etablere primære fibroblastkulturer fra ører og hale i mus. Protokollen kan brukes til å etablere primære fibroblastkulturer fra ørene som er lagret ved romtemperatur i opp til 10 dager. Når protokollen blir fulgt nøye, forurensninger er lite sannsynlig å oppstå til tross for bruk av ikke-sterilt vev lagres i lengre tid i noen tilfeller. Fibroblaster sprer seg raskt i kultur og kan utvides til et betydelig antall før under replicative senescence.

Utvinning av fibroblaster fra vev resulterer i en betydelig mengde vevsrester (figur 1). I motsetning til vev rusk, fibroblaster holde vev kultur plastflatene mellom dag 1 og 3 av kultur. Mediet av fibroblastkulturer trygt kan endres på dag tre av kulturen, som skal betydelig redusere nivåene av rusk som er tilstede i kulturen (figur 2). Fibroblaster viser en langstrakt morfologi og en klart synlig cytoplasma (figur 1 og 2). Mitotiske celler bør vær...

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 rask eksperimentelle fremgangsmåte for å generere primære fibroblaster fra ører og hale i mus. Fremgangsmåten krever ikke spesiell opplæring dyr, og kan brukes for generering av fibroblastkulturer fra ørene som er lagret ved romtemperatur i opp til 10 dager.

Generering Primær fibroblastkulturer fra Muse øre og hale Vev

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