Name: separation of spermatogenic cell types using sta-put velocity sedimentation
Uploaded: 2013-10-09T21:20:00
Description: Watch this Scientific Journal Video about Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation at JoVE.com

Denne forskning blev støttet af NIH tilskud GM055360 SLB og U54HD068157 til RGM. JMB blev støttet af T32 Genetics Training Grant ved University of Pennsylvania (GM008216).

STA-PUT-protokollen omfatter tre faser: 1) Opsætning af apparatet og reagenser, 2) Forberedelse af cellesuspension fra hele testikler, og 3) Cell lastning, sedimentering, og fraktion samling. Når de udføres af et team af to forskere, protokollen tager otte timer i gennemsnit. 1.. Opsætning af STA-PUT Apparatur (figur 1) *** Apparat STA-PUT bør placeres i et 4 ° C stort køleskab eller et koldt rum, der også kan rumme en brøkdel opkøber, hvis denne metode t...

<ol>
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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>Romrell, L. J., Bellve, A. R., Fawcett, 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.+A+morphological+characterization.">Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.</a> Dev. Biol. 49, 119-131 (1976).</li><li>Miller, R. G., Phillips, R. A. <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+cells+by+velocity+sedimentation.">Separation of cells by velocity sedimentation.</a> J. Cell Physiol. 73, 191-201 (1969).</li><li>Bastos, 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=Flow+cytometric+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65, 40-49 (2005).</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>Grabske, R. J., Lake, S., Gledhill, B. L., 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=Centrifugal+elutriation:+separation+of+spermatogenic+cells+on+the+basis+of+sedimentation+velocity.">Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.</a> J. Cell Physiol. 86, 177-189 (1975).</li><li>Meistrich, M. L., Trostle, P. K. <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+testis+cells+by+equilibrium+density+centrifugation+in+renografin+gradients.">Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients.</a> Exp. Cell Res. 92, 231-244 (1975).</li><li>Gassei, K., Ehmcke, J., Schlatt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+enrichment+of+undifferentiated+GFR+alpha+1++spermatogonia+from+immature+rat+testis+by+magnetic+activated+cell+sorting.">Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting.</a> Cell Tissue Res. 337, 177-183 (2009).</li><li>Bellve, A. R., 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=Spermatogenic+cells+of+the+prepuberal+mouse.+Isolation+and+morphological+characterization.">Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization.</a> J. Cell Biol. 74, 68-85 (1977).</li><li>Handel, M. A., Schimenti, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+mammalian+meiosis:+regulation,+dynamics+and+impact+on+fertility.">Genetics of mammalian meiosis: regulation, dynamics and impact on fertility.</a> Nat. Rev. Genet. 11, 124-136 (2010).</li><li>Zhao, M., Shirley, C. R., Mounsey, S., 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=Nucleoprotein+transitions+during+spermiogenesis+in+mice+with+transition+nuclear+protein+Tnp1+and+Tnp2+mutations.">Nucleoprotein transitions during spermiogenesis in mice with transition nuclear protein Tnp1 and Tnp2 mutations.</a> Biol. Reprod. 71, 1016-1025 (2004).</li><li>Manku, G., Mazer, M., Culty, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+testicular+gonocytes+isolation+and+processing+for+immunocytochemical+analysis.">Neonatal testicular gonocytes isolation and processing for immunocytochemical analysis.</a> Methods Mol. Biol. 825, 17-29 (2012).</li><li>Dehnugara, T., Dhar, S., Rao, M. 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Dem, der studerer spermatogenesen stole på dyremodeller for sædcelledannende celleprøver, som en pålidelig cellekultur-system endnu ikke eksisterer for at generere alle sædcelledannende celletyper 3. Selv sædcelledannende celler let indsamles fra hele testikler, kun en blandet population resultat. Det er et problem for dem, der ønsker at studere specifikke undertyper af disse celler, såsom meiotiske celler, runde spermatider og kondenserende spermatider. Er i øjeblikket anvendes tre forskellige metod...

Mammalian spermatogenesen er en kompleks differentiering proces, der foregår i flere trin i sædkanalerne af testiklerne 1. Kort fortalt, stængel-lignende spermatogoner at opholde sig nær epitel sædkanalerne kløft og differentiere i spermatocytter, som derefter undergår meiotiske delinger. Efter meiose er færdig, de resulterende haploide celler eller runde spermatider undergår spermiogenesis, en differentiering proces, der involverer udgydelse af cytoplasma og komprimering af kernen. Spermatider gradvist at udvikle en flagellum og gennemgå forlængelse og kondensering af kernen, der producerer forlængede og derefter kondenserende spermatider, hhv. Slutprodukterne spermatozoer, som frigives ind i lumen af ​​sædkanalerne og til sidst i bitestiklen, hvor de modnes yderligere. Fordi processen med spermatogenesen bygger på særlige hormonelle og molekylære forhold itestiklerne, en pålidelig in vitro-dyrkningssystem for hele processen af spermatogenesen er endnu ikke blevet udviklet 2,3. Er blevet udviklet dyrkningsmetoder for at skabe &quot;primordial kønsceller-lignende celler&quot; og haploide, &quot;runde sædcelleantal-lignende celler&quot; fra stamceller, men disse metoder er endnu ikke i stand til at generere et stort antal af disse celler og undlader at producere senere modningshæmning celle typer 4,5. Heldigvis adskiller de sædcelledannende celletyper betydeligt i størrelse, hvilket giver mulighed for en enkelt-cellesuspension opnået fra hele testiklerne til at blive adskilt med en væske-gradient. STA-PUT metode, viste her, bruger en lineær BSA gradient og enkel sedimentering at adskille sædcelledannende celler baseret på størrelse og masse 6-9. STA-PUT metode har flere fordele i forhold til de andre to mest udbredte metoder til at adskille sædcelledannende celletyper: FACS og slæmning 10-13. Apparat STA-PUT kræver only flere stykker specialiserede glasvarer samlet i et koldt rum eller stort køleskab. Således er det er billigere end anvendelse af et cellesorteringsapparat eller elutriator. STA-PUT metode giver højere mængder af celler pr celletype og testis end kan sorteres ved FACS i en sammenlignelig tidsramme, men renheden af hver cellepopulation er ikke så høje som dem, der opnås med FACS 11. Cellesortering udnytte magnetiske perler (magnetisk aktiveret celle sortering, MACS) er for nylig blevet anvendt med succes til berigelse af spermatogonier fra en blandet testikelkræft celle befolkning, men det er i øjeblikket uegnet til at adskille spermatocytter eller spermatider på grund af manglende viden om hensigtsmæssig overflademarkører 14. En yderligere fordel af STA-metoden PUT i FACS eller MACS er evnen til at isolere levedygtige celler er egnede til efterfølgende kultur, fordi, i modsætning til de fleste FACS-protokoller, kræver det ikke nogen DNA eller andre former for farvning. For undersøgelser, der kræver large udbytter af sædcelledannende celletyper på ~ 90% renhed, STA-PUT er en ideel metode.

<table border="0" cellpadding="0" cellspacing="0" width="856">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">BSA</td>
			<td style="width:109px;">Affymetrix/USB</td>
			<td style="width:255px;">10857 100MG</td>
			<td style="width:183px;">Alternate can be used.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:311px;">0.5%, 2%, and 4% BSA solutions</td>
			<td colspan="2" style="width:364px;">Dissolve 0.25 g, 11 g, and 22 g (respectively) in total volumes of 50 ml, 550 ml, and 550 ml of 1x KREBS (respectively).</td>
			<td style="width:183px;">To be made day of the STA-PUT procedure. Filter sterilize.</td>
		</tr>
		<tr height="91">
			<td height="91" style="height:91px;width:311px;">KREBS (10x)</td>
			<td colspan="2" style="width:364px;">3.26 g KH2PO4 + 139.5 g NaCl +5.89 g MgSO4/7H20 + 40 g Dextrose + 3.78 g CaCl2/2H20 + 7.12 g KCl, bring to 2 L with ddH20</td>
			<td style="width:183px;">Autoclave/filter and store at 4 &#176;C for several months.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:311px;">KREBS (1x)</td>
			<td colspan="2" style="width:364px;">Dissolve 4.24 g NaHCO3 in 100 ml ddH20. Add 200 ml 10x KREBS. Bring to 2 L with ddH20.</td>
			<td style="width:183px;">To be made day of the STA-PUT procedure. Filter sterilize.</td>
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">Collagenase</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">C9891-1G</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">DNAse</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">DNEP-5MG</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">Trypsin</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">T9201-1G</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">DAPI</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Triton-X100</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Formaldehyde (~37%)</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">TABLE 2: EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Equipment</td>
			<td style="width:109px;">Company</td>
			<td style="width:255px;">Catalog #</td>
			<td style="width:183px;">Comments</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:311px;">Complete STA-PUT apparatus</td>
			<td style="width:109px;">ProScience Glass Shop</td>
			<td style="width:255px;">STA-PUT (this procedure uses the standard sedimentation chamber: Cat. No. 56700-500)</td>
			<td style="width:183px;">Includes all glassware and equipment for the apparatus. Alternate glassware is available for performing STA-PUT with smaller cell numbers.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">10 &#956;m mesh filter</td>
			<td style="width:109px;">Fisherbrand</td>
			<td style="width:255px;">22363549</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Mouse dissection tools</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:311px;">14 ml round bottom fraction collection tubes with caps</td>
			<td colspan="3" style="width:547px;">Preference of researcher </td>
		</tr>
		<tr height="22">
			<td colspan="3" height="22" style="height:22px;width:547px;">Need approximately 100 tubes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:311px;">Fraction collector</td>
			<td style="width:109px;">GE Healthcare Life Sciences</td>
			<td style="width:255px;">Model: Frac-920, Product code: 18-1177-40</td>
			<td style="width:183px;">Alternate can be used.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Microscope slides, cover glass</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Light/Fluorescent Microscope</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
	</tbody>
</table>

separation of spermatogenic cell types using sta-put velocity sedimentation

Mammalian spermatogenesen er en kompleks differentiering proces, der foregår i flere trin i sædkanalerne af testiklerne. I øjeblikket er der ingen pålidelige cellekultur-system giver mulighed for modningshæmning differentiering in vitro, og de ​​fleste biologiske studier af sædcelledannende celler kræver væv høst fra dyremodeller som mus og rotter. Fordi testiklerne indeholder mange celletyper - både ikke-spermatogene (Leydig, Sertoli, myeloide og epitelceller) og sædcelledannende (spermatogonier, spermatocytter, runde spermatider, kondenserende spermatider og spermatozoer) - undersøgelser af de biologiske mekanismer, der er involveret i spermatogenesen kræver isolation og berigelse af disse forskellige celletyper. STA-metoden PUT muliggør separation af en heterogen population af celler - i dette tilfælde fra testiklerne - via en lineær BSA gradient. Individuelle celletyper sediment med forskellige sedimentering hastighed i henhold til CEll størrelse, samt fraktioner beriget til forskellige celletyper kan opsamles og anvendes i yderligere analyser. Mens STA-metoden PUT ikke resulterer i meget rene fraktioner af celletyper, fx som kan opnås med visse cellesortering metoder, det giver et meget højere udbytte af totale celler i hver fraktion (~ 1 x 10 8 celler / modningshæmning celletype fra en start befolkning på 7-8 x 10 8 celler). Denne højt udbytte metode kræver kun specialiseret glas og kan udføres i alle kølerum eller stort køleskab, hvilket gør det en ideel metode til laboratorier, som har begrænset adgang til specialiseret udstyr som en fluorescens cellesorteringsapparat (FACS) eller elutriator.

Det ideelle resultat af STA-PUT procedure er en temmelig mærkbar adskillelse af celler fra testiklerne baseret på cellestørrelse og massefylde. Mens celler isoleret fra testiklerne er sedimenting gennem BSA gradient, kan flere forskellige bånd af celler overholdes. Eventuelle klumper af celler har tendens til at synke til bunden af ​​gradienten og vil ikke forurene de øvrige fraktioner. Lidt længere oppe gradienten vil være den store somatiske og meiotiske celler. Længere op gradienten stadig vil være mindr...

Watch this Scientific Journal Video about Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation at JoVE.com

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

STA-metoden PUT tillader adskillelse af forskellige populationer af spermatogene celler baseret på størrelse og tæthed.

Adskillelse af modningshæmning Cell Typer Brug STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

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