이 작품은 독일 Forschungsgemeinschaft (, KL 1415/3-2와 KL 1415/4-2 DFG)에 의해 지원되었다.

1. 준비하기 <ol><li> 배양 접시를 준비 <ol><li> 해동 콜라겐 유형 4에서 IV O / N ° C와 0.1 % 멸균 아세트산에 용해. 사용하는 볼륨은 세포가 파종을 작성할 수있는 요리의 종류와 수에 따라 달라집니다. 2 ㎍ / cm 2를 사용하십시오. </li><li> 적어도 RT에서 1 시간 동안 요리를 품어 증류수 (A. tridest)로 두 번 씻는다. </li><li> 요리가 제대로 건조하도록 허용합니다. </li></ol></li><li> 매체의 보완 <ol><li> 500 ML 매체에 1.75 g의 포도당을 추가하여 최대 4.5 g / L의 포도당 수준을 높일 수 있습니다. 매체 600 mosmol으로 조정하려면, 각각 200 mosmol 100 mosmol하여 삼투압을 증가, 100 mM의 NaCl을 100 MM의 요소를 추가합니다. </li><li> 600 mosmol DMEM 배지에 1 % 비 필수 아미노산, 1 % ultroser, 500 μM의 DBcAMP, 20 U / ㎖ 스타틴 0.25 ㎍ / ㎖ 겐타 마이신 (50 ㎍ / ㎖의 농도 원액 1:200 희석)을 추가 갓 준비의 날에. </li></ol></리&gt; <li> 효소 솔루션 <ol><li> 에 1 밀리그램 / ML 히알루 2.2 MG / ML 콜라를 추가 DPBS 함유 0.25 ㎍ / ㎖의 겐타 마이신과 스타틴을. 2 신장 내부 medullae의 소화에 대한이 효소 용액 1 ㎖를 준비합니다. 이 솔루션은 여과 멸균되어야하며, 50 ML 팔콘 튜브에 저장됩니다. </li></ol></li></ol> 2. 차 쥐 내부 수질의 재배 덕트 (IMCD) 세포를 수집 모든 동물의 처리 절차는 로컬 및 연방 법률을 준수 실시하여야한다. <ol><li> CO 2의 쥐 (CO 2로 포화 상자) 또는 isoflurane을 (; ~ 2.5 % isoflurane을 가진 공기 흐름 350-400 ML / 분 U-400 마취 장치, Univentor 회사, 몰타)을 마취시키다. 흰 안쪽과 어두운 바깥 수질 경계의 정확한 검출을 허용 출혈 동물의 목을 베십시오. 또한, 출혈 격리 적혈구의 수를 최소화합니다. 신장을 제거하고얼음 겐타 마이신과 스타틴 (위 참조) 보충 멸균 DPBS에서 그들을 씻어. 모든 추가 단계는 무균 클린 벤치 조건 하에서 실시하여야한다. </li><li> 과도한 액체를 제거하는 살균 압축에 DPBS 용액으로부터 신장을 전송합니다. 멸균 집게와 가위로 지방 신장 캡슐을 제거합니다. </li><li> 신장은 여전히​​ 압축에 고정 바깥 쪽 (피질) 측의 종축 옆에 약간 잘라 데. 완전히 극복 할 수 있지만, 신장 골반에 연결 한 조각에 남기지 않도록해야합니다. </li><li> 곡선 가위로 조심스럽게 장미 반짝이는 외부 수질에 둘러싸여 흰 내부 medullae를 해부. 얼음에 60mm 배양 접시에 겐타 마이신과 스타틴을 포함 DPBS에서 그들을 수집합니다. </li><li> 모든 medullae이 고립 얼음에 수집되면, 그들은 그냥 버퍼로 덮여 수준으로 DPBS의 양을 줄일 수 있습니다. 5 큐브로 medullae를 잘라 살균 면도칼을 사용하여mm 3. </li><li> 화염으로 짧게 잡고 녹여 멸균 파스퇴르 피펫의 예리한 끝을 라운드. 피펫 계속하기 전에 냉각되어 있는지 확인합니다. </li><li> 둥근 파스퇴르 피펫을 사용하여 히알루 및 콜라게나 제 (1.3.1 참조) 갓 준비한 살균 효소 솔루션을 포함하는 50 ML 팔콘 튜브에 조직 현탁액을 전송합니다. 단단히 뚜껑을 닫고 37 ° C 1.5 일반 탁상 물 목욕 연속 교반 (220 rpm)가 아​​래에서 알을 품다 - 2 시간. </li><li> 서스펜션의 분쇄를 들어, 정지 균일 혼탁 될 때까지 라운드 파스퇴르 피펫 (위 참조)를 여러 번 내려 혼합물을 피펫. </li><li> 300 XG에서 5 분, 16 ° C.를위한 현탁액을 원심 분리기 상층 액을 버리고 침전물을 다시 겐타 마이신과 스타틴과 원심 분리기를 포함 DPBS에 철저 resuspend을. 한번이 세척 단계를 반복합니다. </li><li> 완벽하게 보완 매체와 C에 씨앗을에서 세포를 resuspend을ulture 요리 및 / 또는 미세 역가 웰 플레이트. 2 medullae (1 동물)의 준비를 한 60mm 배양 접시에 충분한 세포를 얻을 것입니다. </li><li> 24 시간 후, 600 mosmol DMEM로 두 번 세포를 씻으 신선한 매체를 추가합니다. 다음 주에 세포를 2 ~ 3 회 (그림 1) 세척해야한다. 6-8일 파종 후 IMCD 세포 실험에 사용할 수 있습니다. 실험을 시작하기 전에 24 시간 동안 DBcAMP 및 스타틴없이 매체를 배양해야합니다. 그렇지 않으면 AQP2는 플라즈마 막에서 독점적 위치합니다. </li></ol>

<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. C., Nairn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+human+cell+line+transformed+by+DNA+from+human+adenovirus+type+5.">Characteristics of a human cell line transformed by DNA from human adenovirus type 5.</a> The Journal of general virology. 36, 59-74 (1977).</li><li>Gluzman, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SV40-transformed+simian+cells+support+the+replication+of+early+SV40+mutants.">SV40-transformed simian cells support the replication of early SV40 mutants.</a> Cell. 23, 175-182 (1981).</li><li>Ala, Y., 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=Functional+studies+of+twelve+mutant+V2+vasopressin+receptors+related+to+nephrogenic+diabetes+insipidus:+molecular+basis+of+a+mild+clinical+phenotype.">Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus: molecular basis of a mild clinical phenotype.</a> Journal of the American Society of Nephrology: JASN. 9, 1861-1872 (1998).</li><li>Richardson, J. C., Scalera, V., Simmons, N. L. <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+two+strains+of+MDCK+cells+which+resemble+separate+nephron+tubule+segments.">Identification of two strains of MDCK cells which resemble separate nephron tubule segments.</a> Biochimica et Biophysica Acta. 673, 26-36 (1981).</li><li>Chen, Y., 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=Aquaporin+2+Promotes+Cell+Migration+and+Epithelial+Morphogenesis.">Aquaporin 2 Promotes Cell Migration and Epithelial Morphogenesis.</a> Journal of the American Society of Nephrology: JASN. , (2012).</li><li>Perantoni, A., Berman, J. 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. R., Weaver, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+characteristics+of+a+pig+kidney+cell+strain,+LLC-PK.">The origin and characteristics of a pig kidney cell strain, LLC-PK.</a> In vitro. 12, 670-677 (1976).</li><li>Nedvetsky, P. I., 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=Reciprocal+regulation+of+aquaporin-2+abundance+and+degradation+by+protein+kinase+A+and+p38-MAP+kinase.">Reciprocal regulation of aquaporin-2 abundance and degradation by protein kinase A and p38-MAP kinase.</a> J. Am. Soc. Nephrol. 21, 1645-1656 (2010).</li><li>Iolascon, 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=Characterization+of+Two+Novel+Missense+Mutations+in+the+AQP2+Gene+Causing+Nephrogenic+Diabetes+Insipidus.">Characterization of Two Novel Missense Mutations in the AQP2 Gene Causing Nephrogenic Diabetes Insipidus.</a> Nephron Physiology. 105, p33-p41 (2007).</li><li>Deen, P. M., 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=Aquaporin-2+transfection+of+Madin-Darby+canine+kidney+cells+reconstitutes+vasopressin-regulated+transcellular+osmotic+water+transport.">Aquaporin-2 transfection of Madin-Darby canine kidney cells reconstitutes vasopressin-regulated transcellular osmotic water transport.</a> Journal of the American Society of Nephrology: JASN. 8, 1493-1501 (1997).</li><li>Valenti, G., Frigeri, A., Ronco, P. M., D'Ettorre, C., Svelto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+functional+analysis+of+water+channels+in+a+stably+AQP2-transfected+human+collecting+duct+cell+line.">Expression and functional analysis of water channels in a stably AQP2-transfected human collecting duct cell line.</a> The Journal of Biological Chemistry. 271, 24365-24370 (1996).</li><li>Steele, S. 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=Telomerase+immortalization+of+principal+cells+from+mouse+collecting+duct.">Telomerase immortalization of principal cells from mouse collecting duct.</a> American Journal of Physiology. Renal Physiology. 299, F1507-F1514 (2010).</li><li>Bens, M., 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=Corticosteroid-dependent+sodium+transport+in+a+novel+immortalized+mouse+collecting+duct+principal+cell+line.">Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.</a> Journal of the American Society of Nephrology: JASN. 10, 923-934 (1999).</li><li>Hasler, U., 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=Long+term+regulation+of+aquaporin-2+expression+in+vasopressin-responsive+renal+collecting+duct+principal+cells.">Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells.</a> The Journal of Biological Chemistry. 277, 10379-10386 (1074).</li><li>Miller, R. L., Sandoval, P. 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. Cell Sci. 116, 3285-3294 (2003).</li><li>Maric, 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=Cell+volume+kinetics+of+adherent+epithelial+cells+measured+by+laser+scanning+reflection+microscopy:+determination+of+water+permeability+changes+of+renal+principal+cells.">Cell volume kinetics of adherent epithelial cells measured by laser scanning reflection microscopy: determination of water permeability changes of renal principal cells.</a> Biophys. J. 80, 1783-1790 (2001).</li><li>Gonzalez, A. 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=Angiotensin+II+stimulates+renin+in+inner+medullary+collecting+duct+cells+via+protein+kinase+C+and+independent+of+epithelial+sodium+channel+and+mineralocorticoid+receptor+activity.">Angiotensin II stimulates renin in inner medullary collecting duct cells via protein kinase C and independent of epithelial sodium channel and mineralocorticoid receptor activity.</a> Hypertension. 57, 594-599 (2011).</li><li>Chou, C. 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=Regulation+of+aquaporin-2+trafficking+by+vasopressin+in+the+renal+collecting+duct.+Roles+of+ryanodine-sensitive+Ca2++stores+and+calmodulin.">Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Ca2+ stores and calmodulin.</a> The Journal of Biological Chemistry. 275, 36839-36846 (2000).</li><li>Uawithya, P., Pisitkun, T., Ruttenberg, B. E., Knepper, M. 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. Am. Soc. Nephrol. 18, 199-212 (2007).</li><li>Klussmann, 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=An+inhibitory+role+of+Rho+in+the+vasopressin-mediated+translocation+of+aquaporin-2+into+cell+membranes+of+renal+principal+cells.">An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.</a> J. Biol. Chem. 276, 20451-20457 (2001).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

우리는 차 쥐 IMCD 세포의 준비 및 배양에 대한 자세한 프로토콜을 제시한다. 방법은 하나의 쥐 20의 세포의 21cm 2까지 얻을 수 있습니다. 실험은 표준 세포 배양 장비가 필요하고 약 6 시간 안에 한 사람에 의해 수행 할 수 있습니다. 따라서이 방법은 표준 실험 방법으로 적합합니다. 차 쥐 IMCD 세포를 96 웰 플레이트에서 60mm 요리에 이르기까지 다양한 크기의 배?...

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

신장 집합관 주 세포, 아르기닌 바소프레신​​ (AVP)는 물 삽입을 자극하여 물을 재 흡수를 제어하는​​ 플라즈마 막에 채널 아쿠아 포린-2 (AQP2). AVP는 아데 cyclase의함으로써 캠프의 형성을 자극 G 단백질 결합 바소프레신​​ 2 형 수용체 (V2R)에 바인딩합니다. 이 신호 폭포의 개시 단백질 키나제 A (PKA)의 활성화로 연결됩니다. PKA는 플라즈마 막으로 세포 내 소포에서의 재분배를위한 주요 트리거입니다, 256 (S256) 세린에서 AQP2를 인산화. 막 삽입 삼투 기울기 및 미세 조정 몸 물 항상성을 따라 물을 재 흡수를 용이하게합니다. 때문에 AVP 분비 또는 AVP 활성화 신호 원인 또는 수차에 AVP-매개 물의 재 흡수의 조절 장애는 심각한 질병과 연관됩니다. V2R 또는 AQP2의 돌연변이에 의해 발생 감소 물을 재 흡수는 표고 반면, 신원 성 요붕증로 연결ated 혈장 AVP 레벨은 만성 심장 마비와 부적절한 항 이뇨 호르몬 분비 (SIADH)의 증후군 심혈관 질환의 과도한 수분 재 흡수와 연결되어 있습니다. AVP-매개 물을 재 흡수의 중요성뿐만 아니라 질병의 의미에서 유래한다. AQP2 베어링에 소포와 세포막과 융합의 AVP 유발 전위는 현재 잘 이해되지 않는 엄격 캠프에 의존 exocytic 프로세스를 나타냅니다. 캠프 의존 exocytosis에 대한 다른 예는 신장과 H의 레닌 분비 + 위장 분비한다. 따라서 신장 주요 세포에서 AQP2-전위를 지배하는 분자 메커니즘의 해명뿐만 아니라 다른 세포 유형에 비슷한 분자 프로세스를 이해하는 데 도움이뿐만 아니라 AVP-매개 물을 재 흡수의 장애와 관련된 질환의 치료에 대한 새로운 치료에 방법을 포장 수 있습니다 . E기계 제어 AQP2를 lucidating은 덕트 주요 세포 모델을 수집해야합니다. 이를 위해 다른 포유 동물의 신장 세포 라인의 숫자를 사용할 수 있습니다. 그러나 이러한 모델은 몇 가지 단점을 가지고있다. 많은 세포 시스템에서 AQP2의 단백질 수준 (표 1, M1, HEK293, COS-7, MDCK, LLC-PK1) 낮은 1-10 ectopically 다른 과발현 사람 (MCD4, WT10) 11,12 또는 쥐 (CD8) 13 AQP2. MCD4 및 COS​​-7 세포에서 V2 수용체가 표시되지 않습니다. 우리의 지식이 신장 내 수질 집합관, AQP2 발현이 가장 풍부한 신장의 그 부분에서 파생 가능한 모델로 영구 세포 라인은 없지만, 대뇌 피질 집합관 1,11,13-16에서 . 이러한 세포 모델은 예를 들어 널리 사용되는 불후의 mpkCCD (예를 들어, 17) 또는 최근에 설립 된 mTERT-CCD 14 세포주이다. 두 세포주 내생 적 표현 AQP2 및 V2R 그러나 그들은에서 파생되기 때문에대뇌 피질의 수집 덕트는 대부분 덕트 (IMCD) 세포를 수집하는 내부 수질에 비해 다른 프로테옴을 포함합니다. 그들은 예를 들어 다른 나 표현해야 + 교통 시스템입니다. 여기, 우리는 V2R 및 AQP2 내생 적 표현 문화의 기본 IMCD 세포 프로토콜을 제시한다. 이 모델은, 그러므로, 신장 수집 덕트에서 가장 밀접하게 생리적 상황을 나타냅니다. 문화를 확립하는 것이 필요하기는 표준 실험실 장비 기타 실험실은 쉽게이 방법을 채택 할 수 있습니다.

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			<td>Name </td>
			<td>Company Catalog</td>
			<td>Number</td>
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		<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>
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		<tr>
			<td>Collagenase type CLS-II</td>
			<td>Biochrom AG</td>
			<td>C2-22</td>
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			<td>DMEM + GlutMAX</td>
			<td>Invitrogen GIBCO</td>
			<td>21885108</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>SIGMA</td>
			<td>N4014</td>
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			<td>Ultroser G</td>
			<td>Cytogen</td>
			<td>15950-017</td>
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			<td>Non essential amino acids (NEA)</td>
			<td>Biochrom AG</td>
			<td>K0293</td>
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			<td>Gentamicin</td>
			<td>Invitrogen GIBCO</td>
			<td>15710</td>
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			<td>DBcAMP</td>
			<td>BIOLOG</td>
			<td>D009</td>
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culturing primary rat inner medullary collecting duct cells

아르기닌 바소프레신​​ (AVP)는 신장 덕트 주요 세포를 수집하고이를 미세 조정 몸 물 항상성에 의해 물을 재 흡수를 용이하게합니다. AVP는 세포의 표면에 바소프레신​​ V2 수용체 (V2R)에 결합함으로써 캠프의 합성을 유도합니다. 이 물 채널 아쿠아 포린-2 (AQP2)의 인산화의 변화를 선도하는 세포 신호 전달 과정을 자극한다. 단백질 키나아제 A는 AQP2를 phoshorylates함으로써 기본 소변에서 물을 재 흡수를 촉진 플라즈마 막으로 세포 내 소포에서 AQP2의 전위를 트리거합니다. 주요 세포에서 뇌하수체 또는 AVP 활성화 신호에서 AVP 방출의 수차는 각각 중앙 또는 신원 성 요붕증을 일으킬 수 있으며, 높은 혈액 혈장 AVP 레벨은 만성 심장 마비와 부적절한 항 이뇨 호르몬 분비 증후군 심혈관 질환과 관련이 있습니다. 여기, 우리는 cultivati​​위한 프로토콜을 제시한다표현 V2R 및 AQP2는 내생 덕트 (IMCD) 세포를 수집, 차 쥐 내부 수질의합니다. 세포는 AQP2의 컨트롤을 기본 분자 메커니즘을 해명에 적합하며, 따라서 AVP-매개 물의 재 흡수의 조절 장애와 관련된 질환의 치료를위한 새로운 약물 표적을 발견 할 수 있습니다. IMCD 세포는 쥐의 신장 내 medullae에서 얻을 수 있습니다 및 6~8일 파종 후 실험에 사용됩니다. IMCD 세포는 일반 세포 배양 접시, 플라스크 및 다른 형식의 마이크로 타이 터 플레이트에서 배양 할 수있는 절차는 몇 시간이 필요하며, 표준 세포 배양 실험실에 적합합니다.

차 쥐 IMCD 세포의 성공적인 재배 6-8일 파종 후 합류 단층 (그림 2)가 발생합니다. 60mm 배양 접시 당 약 6 × 10 6 세포가 있습니다. 이들은 콜라겐 유형 IV, 기저막 구성 요소를 18로 코팅 된 것과 세포는 단단히 배양 접시에 부착. 따라서 IMCD 세포는 여러 철저한 세척 과정에서도 분리되지 않습니다. 까지 배양 세포의 80 %는 내생 V2R 및 AQP2를 표현한다. AQP2가 부족한 세포가 협...

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

아르기닌 바소프레신​​ (AVP)는 신장 주요 세포에 의해 물을 재 흡수를 촉진을 통해 몸에 물 항상성의 미세 조정을 제어합니다. 여기, 우리는 AVP-매개 물 재 흡수를 기본 분자 메커니즘의 해명에 적합한 덕트 세포를 수집 차 쥐 내부 수질의 재배를위한 프로토콜을 제시한다.

덕트 세포를 수집 배양 차 쥐 내부 수질

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

culturing-primary-rat-inner-medullary-collecting-duct-cells

Research

JoVE Journal

Biology

14.7K 조회수.  Max-Delbrück-Center for Molecular Medicine. 아르기닌 바소프레신​​ (AVP)는 신장 주요 세포에 의해 물을 재 흡수를 촉진을 통해 몸에 물 항상성의 미세 조정을 제어합니다. 여기, 우리는 AVP-매개 물 재 흡수를 기본 분자 메커니즘의 해명에 적합한 덕트 세포를 수집 차 쥐 내부 수질의 재배를위한 프로토콜을 제시한다.

비디오: Culturing Primary Rat Inner Medullary Collecting Duct Cells

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

외과 듀엣으로 Nociceptive 및 염증성 중재자를 수집 및 측정

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

연구

생물학

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

쥐 우수한 자궁암 신경 (SCG)에서 Culturing 교감 신경에 대한 프로토콜

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

성인 쥐 심장 Myocytes의 차 문화

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Explants에서 들어라 기본 인간 기관지 상피 세포

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

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. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

마우스 기관에서 자연적으로 흐르는 것과 셀 및 Submucosal 글 랜드 덕트 세포의 분리

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. ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

쥐 간장 세포의 분리 및 차 문화

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 chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

일차 종양에서 면역 세포의 분리

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

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

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

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

배아 배양하는 방법<em&gt; C. 엘레</em&gt; 셀

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

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...