Name: isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin
Uploaded: 2017-07-14T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin at JoVE.com

Dette arbejde blev støttet af NATIONAL INSTITUT OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES grant (R01AR069653 til Zhang LJ) og National Institutes of Health Support (5T32AR062496-03 til CA).

Alle dyreforsøg er godkendt af UCSD Institutional Animal Care and Use Committee. 1. Primær mus KC-isolering og kultur fra neonatal hud <ol><li> Opfylde postnatale dag 0-2 nyfødte fra C57B / 6 vildtype musestammen ved afkapning ved anvendelse af saks. Afskær lemmerne lige over håndled og ankelledder, og afskær derefter halen helt og efterlader et lille hul. </li><li> For at fjerne hele huden skal du først indsætte en skarp saks gennem hulet ved halen og skære huden langs kr...

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-Like+Receptor+3+Activation+Is+Required+for+Normal+Skin+Barrier+Repair+Following+UV+Damage.">Toll-Like Receptor 3 Activation Is Required for Normal Skin Barrier Repair Following UV Damage.</a> J Invest Dermatol. 135 (2), 569-578 (2015).</li><li>Borkowski, A. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage-inducible+C-type+lectin,+mincle,+is+an+essential+component+of+the+innate+immune+response+to+Candida+albicans.">The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans.</a> J Immunol. 180 (11), 7404-7413 (2008).</li><li>Vasioukhin, V., Bauer, C., Yin, M., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+actin+polymerization+is+the+driving+force+for+epithelial+cell-cell+adhesion.">Directed actin polymerization is the driving force for epithelial cell-cell adhesion.</a> Cell. 100 (2), 209-219 (2000).</li><li>Zhuang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+receptor+p55+plays+a+pivotal+role+in+murine+keratinocyte+apoptosis+induced+by+ultraviolet+B+irradiation.">TNF receptor p55 plays a pivotal role in murine keratinocyte apoptosis induced by ultraviolet B irradiation.</a> J Immunol. 162 (3), 1440-1447 (1999).</li><li>Lichti, U., Anders, J., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+short-term+culture+of+primary+keratinocytes,+hair+follicle+populations+and+dermal+cells+from+newborn+mice+and+keratinocytes+from+adult+mice+for+in+vitro+analysis+and+for+grafting+to+immunodeficient+mice.">Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice.</a> Nat Protoc. 3 (5), 799-810 (2008).</li><li>Dlugosz, A. A., Glick, A. B., Tennenbaum, T., Weinberg, W. C., Yuspa, S. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+murine+epidermal+differentiation+markers+is+tightly+regulated+by+restricted+extracellular+calcium+concentrations+in+vitro.">Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.</a> J Cell Biol. 109 (3), 1207-1217 (1989).</li><li>Yuspa, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+for+proliferation+and+differentiation+in+keratinocytes.">Signal transduction for proliferation and differentiation in keratinocytes.</a> Ann N Y Acad Sci. 548, 191-196 (1988).</li><li>Fang, N. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+enhances+mouse+keratinocyte+differentiation+in+vitro+to+differentially+regulate+expression+of+papillomavirus+authentic+and+codon+modified+L1+genes.">Calcium enhances mouse keratinocyte differentiation in vitro to differentially regulate expression of papillomavirus authentic and codon modified L1 genes.</a> Virology. 365 (1), 187-197 (2007).</li><li>Kolly, C., Suter, M. M., Muller, E. 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Hudens epidermis fungerer som en kritisk barriere for at adskille og beskytte kroppen udefra og beskadige vandfald, patogener, varme og UV-bestråling. KC&#39;erne er epidermis fremherskende cellelinie, og primære kulturer af epidermale KC&#39;er er et nyttigt redskab til at studere og forstå de biologiske processer af barrieredannelse og KCs reaktion på miljøbelastninger in vitro . Her beskriver vi metoder til at isolere og dyrke primære epidermale KC&#39;er fra neonatal og vo...

Huden er det største organ i kroppen med epidermis som det yderste lag. Den epidermis spiller en afgørende rolle for at danne en intakt epidermal barriere for at adskille kroppen fra miljøet og forhindrer dermed vandtab og giver beskyttelse mod miljøbelastninger, såsom allergener, patogener og UVB-eksponering. Epidermis udvikler sig fra et enkelt lag af udifferentierede basale keratinocytter (KC&#39;er) ind i et flerskiktsstratificeret epitel, der består af et basal lag efterfulgt af et spinous lag, granulært lag og stratum corneum. Basal KC&#39;er, der består af både epidermale stamceller og transiteringsforstærkende celler, er proliferative og udifferentierede. Når basale KC&#39;er forlader cellecyklussen, forpligter cellerne sig til differentiering og gradvist migrere mod overfladen af ​​epidermis, ledsaget af modning af cellecelleforbindelser og dannelse af en epidermal permeabilitetsbarriere (EPB). KC&#39;erne ved spinouslaget udtrykker tidligt differentierIonmarkører såsom Keratin 10 (K10); Når KC&#39;erne migrerer til det granulære lag, udtrykker cellerne sen differentieringsmarkører såsom Filaggrin (FLG), Loricrin (LOR) og Involucrin (INV). Ved stratum corneum bliver KC&#39;erne terminalt differentierede corneocytter, som til sidst udvises ved desquamation, idet nye celler erstatter dem. Calcium betragtes som det mest fysiologiske middel i epidermis og udløser differentiering in vitro og in vivo på en lignende måde. I normale hudepidermier danner calciumioner en karakteristisk &quot;koncentrationsgradient&quot;, der stiger i koncentration mod hudoverfladen 1 , 2 , 3 . Kalciumkoncentrationen stiger fra lave niveauer i de laveste underlag (basale og spinøse lag) til en top i det øvre granulære lag og derefter falder til ubetydelige niveauer i det mest overfladiske lag (stratum corneum).<html> Kalciumgradienten udvikler sig også tilfældigt med fremkomsten af ​​en komponentpermeabilitetsbarriere, hvilket understøtter, at calciumsignaler spiller en kritisk rolle for KC-differentiering. In vitro opretholder lavt calcium (0,02-0,1 mM) proliferationen af ​​basale KC&#39;er som et monolag, hvorimod højt calcium (&gt; 0,1 mM) inducerer en hurtig og irreversibel forpligtelse af cellerne til terminal differentiering som demonstreret ved tæt krydsningsdannelse og induktion Af LOR og INV på høj calciumbehandling til de basale KCs 4 , 5 . Ud over barrieredannelse er epidermale KC&#39;er også en vigtig bestanddel af hudens medfødte immunsystem. Som reaktion på patogener eller beskadigede associerede molekylære mønstre (DAMP&#39;er), der frigives ved UVB-bestråling eller -skade, kan KC&#39;er producere store mængder inflammatoriske cytokiner, såsom TNFa, IL6 og IFNβ, hvilket fører til immunsystemaktivering&gt; 6 , 7 , 8 , 9 . Selvom der kræves korrekt inflammatorisk signalering fra KC&#39;er til patogen clearance, kan ukontrolleret inflammatorisk respons udløse udviklingen af ​​autoinflammatoriske hudsygdomme, såsom psoriasis og rosacea 6 , 8 . Samlet set spiller KC&#39;er en afgørende rolle for at opretholde den intakte hudbarriere og initierer et immunrespons ved patogeninvasion eller miljøbelastninger. Derfor er primærkultur af epidermale KC&#39;er en nyttig teknik til at studere epitelbiologi, KC-differentiering, såvel som KC-stimulerede medfødte immunresponser. Isolering og kultur af primære musepidermuskulære KC&#39;er kan være en udfordrende proces på grund af KCs modtagelighed og følsomhed over for forskellige eksterne stimulanter. Her beskriver vi en metode til at isolere og dyrke KC&#39;er fra enten neonatal museskind ellerVoksen mus hale hud. Til voksen KC-isolering bruger vi ikke musens dorsale hud, fordi isolering af tilstrækkelige mængder levedygtige KC&#39;er fra dette væv kan være svært af følgende grunde: Først består den voksne dorsale hud i hvilepasen af ​​hårcyklusen (telogen) af en Tynde epidermier med kun 1-2 lag celler, hvilket fører til lav celleudbytte og ineffektiv adskillelse af epidermis fra dermis, hvilket er det kritiske trin for succesfuld KC-isolering. For det andet bidrager den høje hårfollikeldensitet, som er til stede på voksen dorsal hud, yderligere til vanskeligheden ved at adskille epidermier fra dermis. I stedet bruger vi rutinemæssigt halehud som kilde til voksne mus-KC&#39;er, da dette epitel er tykkere med 3-5 lag epidermal KCs. Det har også en lavere hårfollikeltæthed, som ikke forstyrrer epidermiseparationen, hvilket tillader KC-isolering fra en hvilken som helst voksen musestikhud uanset musens alder og hårcyklusstadium. Den isolerede neonaTal KC&#39;er er podet til gelatinebelagte kulturskåle, hvorimod kollagenbelagte skåle anvendes til at frø isolerede voksne KC&#39;er på grund af den svækkede evne hos de voksne celler til at klæbe i forhold til deres neonatale modparter. Til dyrkning af mus-KC&#39;er suppleres lavt calciumbasalt medium med dGS, som indeholder epidermal vækstfaktor (EGF), bovint transferrin, insulinlignende vækstfaktor1 (IGF1), prostaglandin E2 (PGE2), bovint serumalbumin (BSA) og hydrocortison. Mellem 2-4 dage efter den oprindelige plettering kan de fleste af de differentierede KC&#39;er vaskes væk under daglige mediumændringer, og de resterende vedhæftende celler viser typisk brostenmorfologi 4 , prolifererer og udtrykker ikke den tidlige differentieringsmarkør K10.

<table width="633">
	<tbody>
		<tr height="100">
			<td height="100" style="width:171px;height:100px;">C57BL/6 neonates or adult wildtype mice</td>
			<td style="width:177px;">Jackson Laboratory</td>
			<td style="width:119px;">000664</td>
			<td style="width:167px;">Wildtype mice (originally purchased from Jackson Laboratory) are breeded and maintained in animal vivarium at UCSD.</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">KC basal medium (EpiLife)</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">MEPICF500</td>
			<td style="width:167px;">basal medium for keratinocyte culture with 0.06 mM CaCl2</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">Defined Growth Supplement (dGS)</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">S0125</td>
			<td style="width:167px;">defined growth supplements for culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Dispase powder</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">17105041</td>
			<td style="width:167px;">enzyme to dissociate the epidermis from dermis</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Attachment Factor</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">S006100</td>
			<td style="width:167px;">gelatin-based coating material</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Coating Matrix</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">R011K</td>
			<td style="width:167px;">Collagen-based coating material</td>
		</tr>
		<tr height="80">
			<td height="80" style="width:171px;height:80px;">TrypLE</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">12604-013</td>
			<td style="width:167px;">A gentle trypsin-like enzyme to dissociate keratinocytes from epidermal sheet</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">100 &#956;m Cell Strainer Nylon mesh</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">352360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">CCK-8 cell viability Kit</td>
			<td style="width:177px;">Dojindo Molecular Technologies, Rockville, MD</td>
			<td style="width:119px;">CK04-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Mouse TNF (Mono/Mono) ELISA Set II</td>
			<td style="width:177px;">BD Biosciences, San Jose, CA</td>
			<td style="width:119px;">555268</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Corded Hand-Held UV Lamps</td>
			<td style="width:177px;">Spectronics, Westbury, NY</td>
			<td style="width:119px;">EB-280C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:171px;height:20px;">8-watt UV tubes</td>
			<td style="width:177px;">Spectronics, Westbury, NY</td>
			<td style="width:119px;">BLE-8T312</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Light Inverted Microscope for cell culture</td>
			<td style="width:177px;">ZEISS, Jena, Germany</td>
			<td style="width:119px;">Axio Observer</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:171px;height:20px;">Fluorescent Microscope</td>
			<td style="width:177px;">Olympus</td>
			<td style="width:119px;">BX41</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin

Keratinocyten (KC) er den overvejende celletype i epidermis, det yderste lag af huden. Epidermal KC&#39;er spiller en afgørende rolle for at yde hudforsvar ved at danne en intakt hudbarriere mod miljøbelastninger, såsom UVB-bestråling eller patogener, og også ved at starte en inflammatorisk reaktion på disse fornærmelser. Her beskriver vi metoder til at isolere KC&#39;er fra neonatal museskind og fra voksen musestikhud. Vi beskriver også dyrkningsbetingelser ved anvendelse af definerede væksttilskud (dGS) i sammenligning med chelexed føtalt bovint serum (cFBS). Funktionelt viser vi, at både neonatale og voksne KC&#39;er er yderst lydhøre over for høj calciuminduseret terminal differentiering, tæt krydsdannelse og stratificering. Derudover er kultiverede voksne KC&#39;er modtagelige for UVB-udløst celledød og kan frigive store mængder TNF ved UVB-bestråling. Sammen vil de her beskrevne metoder være nyttige for forskere til opsætningen af in vitro- modellerS at studere epidermisk biologi i neonatalmusen og / eller den voksne mus.

Højkalciuminduceret terminal differentiering af neonatale og voksne KC&#39;er. Den primære muse epidermale KCS udpladet og opretholdt ved 0,06 mM CaCl2 voksede som et monolag, og individuelle celler havde en polygonal form med tydelig intercellulære rum, der viser en brosten udseende, når sammenflydende (figur 1A og figur 2A). Omring CaCI2 0,2 mM inducerede en hurtig morfologi ændring ...

Watch this Scientific Journal Video about Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin at JoVE.com

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

Epidermal keratinocytter danner en funktionel hudbarriere og er placeret på forsiden af ​​værtsforsvaret mod eksterne miljøbelastninger. Her beskriver vi metoder til isolering og primærkultur af epidermale keratinocytter fra neonatal og voksen museskind og induktion af terminal differentiering og UVB-udløst inflammatorisk respons fra keratinocytter.

Isolering og kultur af primære muskeratinocytter fra neonatal og voksen mus hud

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Isolering og dyrkning af Neonatal Mouse cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Isolation, Kultur og funktionel karakterisering af Voksen Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolering og dyrkning af Mouse Primære Pancreas acinarceller

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Isolering af Primære myofibroblasts fra mus og Human tyktarmsvæv

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Isolering og dyrkning af Dental Epiteliale stamceller fra voksne mus Fortand

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolering og dyrkning af Voksen Mouse kardiomyocytter for cellesignalering og<em&gt; In vitro</em&gt; Hjertehypertrofi

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolering og primær kultur af muse aortaendotelceller

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Isolation, Kultur og transduktion af Voksen Mouse cardiomyocytter

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...