Name: simple and efficient technique for the preparation of testicular cell suspensions
Uploaded: 2013-08-04T14:00:00
Description: Watch this Scientific Journal Video about Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions at JoVE.com

Dette arbejde blev delvist støttet af CSIC (I + U-projekt C022) og PEDECIBA. Forfatterne vil gerne takke Mariela Bollati og Valentina Porro fra Cell Biology Unit (Institut Pasteur de Montevideo) for deres generøse samarbejde vedrørende MoFlo cellesorteringsapparat og Merial-Montevideo for forsigtigt give alle de marsvin prøver, der anvendes i dette projekt. Figur 1 blev gengivet med tilladelse fra BioMed Central, figur 2 og 3, og tabel 1 med tilladelse fra S. Karger AG, Basel, og figur 4 og 5 er gengivet eller tilpasset med tilladelse fra John Wiley &amp; Sons, Inc (ophavsret ejes af Wiley- Blackwell, 2011).

1.. Fremstilling af cellesuspensioner <ol><li> Sacrifice prøven skal anvendes efter anbefalingerne fra fagudvalgene såsom IACUC eller tilsvarende (i Uruguay, Nationale Kommission for dyreforsøg [CNEA]). I vores tilfælde var en overdosis af pentobarbital administreres. </li><li> Dissekere testiklerne efter standard godkendte procedurer og placere dem i en 96 mm glas petriskål på is, som indeholder 10 ml iskold DMEM suppleret med 10% føtalt kalveserum. </li><li> Fjern Tunica albuginea og skær decapsulated testik...

<ol>
	<li>Meistrich, M. L., Prescott, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+spermatogenic+cells+and+nuclei+from+rodent+testes.">Separation of spermatogenic cells and nuclei from rodent testes.</a> Methods of Cell Biology. 15, 15-54 (1977).</li><li>Lam, D. M. K., Furrer, R., Bruce, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+separation,+physical+characterization,+and+differentiation+kinetics+of+spermatogonial+cells+of+the+mouse.">The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse.</a> Proc. Natl. Acad. Sci. USA. 65, 192-199 (1970).</li><li>Romrell, L. J., Bellve, A. R., Fawcet, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.">Separation of mouse spermatogenic cells by sedimentation velocity.</a> Dev. Biol. 19, 119-131 (1976).</li><li>Geisinger, A., Rodr&#237;guez-Casuriaga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+for+gene+expression+studies+in+mammalian+spermatogenesis.">Flow cytometry for gene expression studies in mammalian spermatogenesis.</a> Cytogenet. Genome Res. 128, 46-56 (2010).</li><li>Getun, I. V., Torres, B., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. (50), e2602 (2011).</li><li>Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+velocity+sedimentation.">Separation of mouse spermatogenic cells by velocity sedimentation.</a> J. Cell Physiol. 80, 299-312 (1972).</li><li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Malkov, M., Fisher, Y., Don, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+schedule+of+the+postnatal+rat+testis+determined+by+flow+cytometry.">Developmental schedule of the postnatal rat testis determined by flow cytometry.</a> Biol. Rep. 59, 84-92 (1998).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., Santi&#241;aque, F., L&#243;pez, B., Folle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-purity+flow+sorting+of+early+meiocytes+based+on+DNA+analysis+of+guinea+pig+spermatogenic+cells.">High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.</a> Cytometry A. 79, 625-634 (2011).</li><li>Davey, H. M., Hexley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+but+not+dead?+Membranes+of+stressed+Saccharomyces+cerevisiae+are+permeable+to+propidium+iodide.">Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide.</a> Environ. Microbiol. 13, 163-171 (2011).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., L&#243;pez-Carro, B., Porro, V., Wettstein, R., Folle, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-fast+and+optimized+method+for+the+preparation+of+rodent+testicular+cells+for+flow+cytometric+analysis.">Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.</a> Biol. Proced. Online. 11, 184-195 (2009).</li><li>Span&#242;, M., Evenson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+for+reproductive+biology.">Flow cytometric analysis for reproductive biology.</a> Biol Cell. 78, 53-62 (1993).</li></ol>

Den optimerede her beskrevne metode muliggør fremstillingen af ​​cellesuspensioner fra gnaver testikelvævet i en meget hurtig og reproducerbar måde, der undgår enzym og vaskemiddel behandling og opretholde god celle integritet og type proportioner. Den korte procedure (15 min span omfatter testis dissektion, vævs skæring og forarbejdning), minimal involveret håndtering, og fravær af enzymatiske behandlinger er nogle af de vigtigste fordele. Alle disse ville forklare den gode bevarelse af korte levetid makrom...

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Medimachine system</td>
 <td>BD</td>
 <td>340587</td>
 <td></td>
 </tr>
 <tr>
 <td>Medicon unit (50 &mu;m)</td>
 <td>BD</td>
 <td>340591</td>
 <td></td>
 </tr>
 <tr>
 <td>Filcon unit (50 &mu;m)</td>
 <td>BD</td>
 <td>340603</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>430-2100</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>PAA</td>
 <td>A11-151</td>
 <td></td>
 </tr>
 <tr>
 <td>NDA</td>
 <td>Chemos BmbH</td>
 <td>277081</td>
 <td></td>
 </tr>
 <tr>
 <td>Hoechst 33342</td>
 <td>Sigma-Aldrich</td>
 <td>14533</td>
 <td>Stock solution 5 mg/ml; used at a final concentration of 5 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Propidium iodide</td>
 <td>Sigma-Aldrich</td>
 <td>287075</td>
 <td>Stock solution 1 mg/ml; used at a final concentration of 50 &mu;g/ml</td>
 </tr>
 </tbody>
</table>

simple and efficient technique for the preparation of testicular cell suspensions

Mammale testikler er meget komplekse organer, der indeholder over 30 forskellige celletyper, herunder somatiske testikelceller og forskellige stadier af kønscelleoverførsel celler. Denne uensartethed er en vigtig ulempe vedrørende undersøgelse af grundlaget for pattedyr spermatogenesen, som rene eller beriget celle populationer i visse faser af udviklingen af sæd der er behov for de fleste molekylære analyser 1.. Forskellige strategier såsom Staput 2,3, centrifugal slæmning 1, og flowcytometri (FC) 4,5 er blevet anvendt til at opnå berigede eller oprensede testiklerne cellepopulationer for at muliggøre differential genekspressionsstudier. Det kræves, at cellerne er i suspension fleste berigelse / oprensning tilgange. Ideelt set vil cellesuspensionen være repræsentativ for det oprindelige væv, har en høj andel af levedygtige celler og få multinucleates - som har en tendens til at danne grund afsyncytial karakter seminiferous epitel 6,7 - og mangel celleklumper 1.. Tidligere rapporter havde dokumenteret, at testiklerne cellesuspensioner udarbejdet af en udelukkende mekanisk metode klumpet lettere end trypsinerede dem 1.. På den anden side, behandlinger enzymatiske med RNAser og / eller disaggregering enzymer som trypsin og collagenase føre til specifikke makromolekyler nedbrydning, hvilket er uønsket for visse efterfølgende anvendelser. Den ideelle proces bør være så kort som muligt og involvere minimal manipulation, således at der opnås en god konservering af makromolekyler af interesse såsom mRNA&#39;er. Nuværende protokoller til fremstilling af cellesuspensioner fra faste væv er normalt tidskrævende, stærkt afhængig af operatøren, og kan selektivt beskadige visse celletyper 1,8. Protokollen præsenteres her kombinerer fordelene ved en meget reproducerbar og ultrakort mekanisk disaggregering med enbsence af enzymatisk behandling, hvilket fører til god kvalitet cellesuspensioner, der kan bruges til flowcytometrisk analyse og sortering 4, og skjulte genekspressionsstudier 9..

Et eksempel på et godt disaggregerede cellesuspension fra rotte testikler fremstillet med protokollen beskrevet her er vist i figur 1.. I forhold til enzymatiske behandlinger 6,8 og tidligere beskrevne mekaniske opdelingsniveauer metoder 2, er den præsenteres her meget hurtigere, indebærer mindre håndtering, er let at reproducere (ikke afhænger af operatøren), og gør knappe cellefragment (især sammenlignet med andre mekaniske metoder) og meget f...

Watch this Scientific Journal Video about Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions at JoVE.com

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

En roman protokol for den mekaniske tilberedning af testikelkræft cellesuspensioner fra gnaver materiale, undgå enzymer og rengøringsmidler, der er beskrevet. Fremgangsmåden er meget enkel, hurtig, reproducerbar, og gør god kvalitet cellesuspensioner, som er egnet til flow sortering og RNA-ekstraktion.

Simpel og effektiv teknik til Udarbejdelse af Testikel blodlegemesuspensioner

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

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