Name: culturing primary rat inner medullary collecting duct cells
Uploaded: 2013-06-21T14:00:00
Description: Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

Dette arbejde blev støttet af Deutsche Forschungsgemeinschaft (DFG, KL 1415/3-2 og KL 1415/4-2).

1.. Forberedelse <ol><li> Forberedelse dyrkningsskålene <ol><li> Thaw collagen type IV O / N ved 4 ° C og opløse det i 0,1% steril eddikesyre. Mængden til at bruge, afhænger af typen og antallet af retter, hvor cellerne der skal seedet. Brug 2 ug / cm 2. </li><li> Inkuber retter i mindst 1 time ved stuetemperatur og vaskes to gange med destilleret vand (A. tridest). </li><li> Lad det tørre ordentligt. </li></ol></li><li> Tilskud af medium <ol><li> Øg glukose niveau op til 4,5 g / L ved tilsætning 1,...

<ol>
	<li>Stoos, B. A., Naray-Fejes-Toth, A., Carretero, O. A., Ito, S., Fejes-Toth, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+mouse+cortical+collecting+duct+cell+line.">Characterization of a mouse cortical collecting duct cell line.</a> Kidney International. 39, 1168-1175 (1991).</li><li>Graham, F. L., Smiley, J., Russell, W. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+Wilms'+tumor+line+(TuWi)+and+pig+kidney+line+(LLC-PK1)+typical+of+normal+kidney+tubular+epithelium.">Properties of Wilms' tumor line (TuWi) and pig kidney line (LLC-PK1) typical of normal kidney tubular epithelium.</a> In vitro. 15, 446-454 (1979).</li><li>Katsura, T., 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=Constitutive+and+regulated+membrane+expression+of+aquaporin+1+and+aquaporin+2+water+channels+in+stably+transfected+LLC-PK1+epithelial+cells.">Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 7212-7216 (1995).</li><li>Hull, R. N., Cherry, W. 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C., Pisitkun, T., Knepper, M. A., Hoffert, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasopressin+inhibits+apoptosis+in+renal+collecting+duct+cells.">Vasopressin inhibits apoptosis in renal collecting duct cells.</a> American Journal of Physiology. Renal Physiology. , (2012).</li><li>Kleinman, H. K., 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=Basement+membrane+complexes+with+biological+activity.">Basement membrane complexes with biological activity.</a> Biochemistry. 25, 312-318 (1986).</li><li>Storm, R., Klussmann, E., Geelhaar, A., Rosenthal, W., Maric, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmolality+and+solute+composition+are+strong+regulators+of+AQP2+expression+in+renal+principal+cells.">Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells.</a> American Journal of Physiology. Renal Physiology. 284, 189-198 (2003).</li><li>Maric, K., Oksche, A., Rosenthal, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquaporin-2+expression+in+primary+cultured+rat+inner+medullary+collecting+duct+cells.">Aquaporin-2 expression in primary cultured rat inner medullary collecting duct cells.</a> Am. J. Physiol. 275, 796-801 (1998).</li><li>Liebenhoff, U., Rosenthal, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Rab3-,+Rab5a-+and+synaptobrevin+II-like+proteins+in+a+preparation+of+rat+kidney+vesicles+containing+the+vasopressin-regulated+water+channel.">Identification of Rab3-, Rab5a- and synaptobrevin II-like proteins in a preparation of rat kidney vesicles containing the vasopressin-regulated water channel.</a> FEBS Lett. 365, 209-213 (1995).</li><li>Lorenz, D., 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=Cyclic+AMP+is+sufficient+for+triggering+the+exocytic+recruitment+of+aquaporin-2+in+renal+epithelial+cells.">Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells.</a> EMBO Rep. 4, 88-93 (2003).</li><li>Tamma, G., 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+prostaglandin+E2+analogue+sulprostone+antagonizes+vasopressin-induced+antidiuresis+through+activation+of+Rho.">The prostaglandin E2 analogue sulprostone antagonizes vasopressin-induced antidiuresis through activation of Rho.</a> J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+native+inner+medullary+collecting+duct+cells+from+rat+kidney.">Transcriptional profiling of native inner medullary collecting duct cells from rat kidney.</a> Physiological Genomics. 32, 229-253 (2008).</li><li>Tchapyjnikov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+profiling+of+nuclei+from+native+renal+inner+medullary+collecting+duct+cells+using+LC-MS/MS.">Proteomic profiling of nuclei from native renal inner medullary collecting duct cells using LC-MS/MS.</a> Physiological Genomics. 40, 167-183 (2010).</li><li>Stefan, E., 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=Compartmentalization+of+cAMP-dependent+signaling+by+phosphodiesterase-4D+is+involved+in+the+regulation+of+vasopressin-mediated+water+reabsorption+in+renal+principal+cells.">Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells.</a> J. 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Vi præsenterer en detaljeret protokol for forberedelse og dyrkning af primære rotte IMCD celler. Den tilgang giver op til 21 cm 2 af celler fra en rotte 20. Forsøget kræver standard cellekultur udstyr og kan udføres af en enkelt person inden for ca 6 timer. Derfor er denne fremgangsmåde er egnet som en standard laboratoriemetode. Primære rotte IMCD celler kan podes i dyrkningsskåle af forskellig størrelse fra plader med 96 brønde til 60 mm skåle. Men for væk...

A correction was made to <a href="http://www.jove.com/video/50366">Culturing Primary Rat Inner Medullary Collecting Duct Cells</a>. There was an error with an author's name. The author's last name had a typo and was corrected to: Enno Klussmann instead of: Enno Klussman

A correction to author list has been made for article Culturing Primary Rat Inner Medullary Collecting Duct Cells.

Erratum: Culturing Primary Rat Inner Medullary Collecting Duct Cells

I renale opsamlingskanal vigtigste celler, styrer arginin-vasopressin (AVP) vand reabsorption ved at stimulere indsættelse af vandkanal Aquaporin-2 (AQP2) i plasmamembranen. AVP binder til G-protein-koblede vasopressin type 2-receptoren (V2R) stimulere adenylylcyclase og derved cAMP-dannelse. Indledningen af ​​denne signaleringskaskade fører til aktivering af protein kinase A (PKA). PKA phosphorylerer AQP2 på serin 256 (S256), hvilket er nøglen udløser dens omfordeling fra intracellulære vesikler i plasmamembranen. Membranen indsættelse letter vand reabsorption langs en osmotisk gradient og finjusterer kropsvæske homeostase. Dysregulering af AVP-medieret vand reabsorption, på grund af afvigelser i AVP sekretion eller AVP-aktiverede signal årsager eller forbundet med alvorlige sygdomme. Nedsat vand reabsorption forårsaget af mutationer i V2R eller AQP2 fører til nefrogen diabetes insipidus, mens en Elevated blodplasma AVP niveau er forbundet med overdreven vand reabsorption i cardiovaskulære sygdomme, såsom kronisk hjertesvigt og syndrom med uhensigtsmæssig antidiuretisk hormon (SIADH). Betydningen af ​​AVP-medieret vand reabsorption stammer ikke blot fra dens konsekvenser i sygdom. AVP-induceret translokation af AQP2-bærende vesikler til og fusion med plasmamembranen repræsenterer en strengt cAMP-afhængig eksocytisk proces, som i øjeblikket ikke er godt forstået. Andre eksempler på en cAMP-afhængig exocytose er reninsekretionen i nyrerne og H + sekretion i maven. Derfor udredning af molekylære mekanismer, der styrer AQP2-translokation hos nyre vigtigste celler ikke kun hjælper til at forstå lignende molekylære processer i andre celletyper, men kan også bane vejen for nye behandlingsformer til behandling af sygdomme forbundet med forstyrrelser af AVP-medieret vand reabsorption . Elucidating mekanismer kontrollerer AQP2 kræver opsamlingskanal vigtigste celle modeller. Til dette en række forskellige pattedyr nyrecellelinier er tilgængelige. Men disse modeller har flere ulemper. I mange cellesystemer proteinniveauet af AQP2 er lav (tabel 1, M1, HEK293, COS-7, MDCK, LLC-PK1) 1-10, andre ektopisk overudtrykker humane (MCD4, WT10) 11,12 eller rotte (CD8) 13 AQP2. I MCD4 og COS-7-celler V2-receptoren ikke udtrykkes. Til vores viden er der ingen permanent cellelinie som en model til rådighed, som er afledt af den renale indre medullære opsamlingskanal, at en del af nyren hvor AQP2 ekspression er mest rigelige, men fra den kortikale opsamlingskanal 1,11,13-16 . Sådanne cellemodeller er for eksempel den meget brugte udødeliggjort mpkCCD (fx 17), eller de nyoprettede mTERT-CCD 14 cellelinjer. Begge cellelinjer endogent express AQP2 og V2R men da de er afledt afkortikal opsamlingskanal sandsynligvis indeholde en anden proteom forhold til indre medullær samlekanal (IMCD) celler. De skal for eksempel udtrykker forskellige Na + transportsystemer. Her præsenterer vi en protokol til kultur primære IMCD celler, der udtrykker V2R og AQP2 endogent. Denne model repræsenterer derfor mest nøje den fysiologiske situation i renal opsamlingskanal. Som om kulturen kræver kun Standardlaboratorieudstyr andre laboratorier kan nemt vedtage denne fremgangsmåde.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company Catalog</td>
			<td>Number</td>
		</tr>
		<tr>
			<td>Collagen Type IV Mouse</td>
			<td>BD Biosciences</td>
			<td>356233</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>SIGMA</td>
			<td>H6254</td>
		</tr>
		<tr>
			<td>Collagenase type CLS-II</td>
			<td>Biochrom AG</td>
			<td>C2-22</td>
		</tr>
		<tr>
			<td>DMEM + GlutMAX</td>
			<td>Invitrogen GIBCO</td>
			<td>21885108</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>SIGMA</td>
			<td>N4014</td>
		</tr>
		<tr>
			<td>Ultroser G</td>
			<td>Cytogen</td>
			<td>15950-017</td>
		</tr>
		<tr>
			<td>Non essential amino acids (NEA)</td>
			<td>Biochrom AG</td>
			<td>K0293</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen GIBCO</td>
			<td>15710</td>
		</tr>
		<tr>
			<td>DBcAMP</td>
			<td>BIOLOG</td>
			<td>D009</td>
		</tr>
	</tbody>
</table>

culturing primary rat inner medullary collecting duct cells

Arginin-vasopressin (AVP) letter vand reabsorption ved renal opsamlingskanal vigtigste celler og dermed finjusterer kropsvæske homeostase. AVP binder sig til vasopressin V2-receptorer (V2R) på overfladen af ​​cellerne og derved inducerer syntese af cAMP. Dette stimulerer cellulære signalprocesser der fører til ændringer i phosphorylering af vandkanal Aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 og derved udløser translokation af AQP2 fra intracellulære vesikler i plasmamembranen letter vand reabsorption fra primær urin. Afvigelser i AVP frigivelse fra hypofysen eller AVP-aktiveret signalering i væsentligste celler kan forårsage centrale eller nefrogen diabetes insipidus, henholdsvis en forhøjet blodplasma AVP niveau er forbundet med cardiovaskulære sygdomme, såsom kronisk hjertesvigt og syndrom med uhensigtsmæssig sekretion af antidiuretisk hormon. Her præsenterer vi en protokol for cultivatipå primær rotte indre medullær samlekanal (IMCD) celler, som udtrykker V2R og AQP2 endogent. Cellerne er egnede til at belyse molekylære mekanismer, der ligger kontrol AQP2 og dermed at opdage nye lægemiddelkandidater til behandling af sygdomme forbundet med dysregulering af AVP-medieret vand reabsorption. IMCD celler opnås fra rotte nyre indre medullae og anvendes til eksperimenter seks til otte dage efter podning. IMCD celler kan dyrkes i ordinær celle dyrkningsskåle, kolber og mikro-titer plader af forskellige formater, den procedure, der kun kræver nogle få timer, og er egnet til standard cellekultur laboratorier.

Den vellykkede dyrkning af primære rotte IMCD celler vil resultere i en sammenflydende monolag 6-8 dage efter podning (figur 2). Per 60 mm dyrkningsskål der er ca 6 x 10 6 celler. Cellerne stramt overholde dyrkningsskålene, da disse blev belagt med collagen type IV, en kælder membran komponent 18. Derfor vil IMCD celler ikke frigøre selv i adskillige grundig vask procedurer. Op til 80% af de dyrkede celler udtrykker endogent V2R og AQP2. Disse er de væsentligste celler, mens ...

Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginin-vasopressin (AVP) styrer finjustering af kropsvæske homeostase ved at lette vand reabsorption ved renal vigtigste celler. Her præsenterer vi en protokol til dyrkning af primære rotte indre medullær opsamlingskanal celler egnede til udredning af molekylære mekanismer bag AVP-medieret vand reabsorption.

Dyrkning Primær Rat Inner Medullær samlekanal Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

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Isolering af basale celler og submucosal Gland Duct celler fra mus Luftrøret

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Udarbejdelse af muse embryonale fibroblastceller anvendes til dyrkning af menneskelige embryoner og induceret pluripotente stamceller

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Dyrkning af human Nasal epithelialceller på Air Liquid grænseflade

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

A Method for Culturing Embryonic C. elegans Cells

En fremgangsmåde til dyrkning Embryonale<em&gt; C. elegans</em&gt; Celler

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...