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

这项研究是由美国国立卫生研究院资助GM055360为SLB和U54HD068157到RGM支持。 JMB由T32遗传学培训格兰特在宾夕法尼亚大学（GM008216）的支持。

在STA-PUT协议包括三个阶段：1）设置仪器和试剂，2）准备从整个睾丸细胞悬液，以及3）小区负荷，沉淀和馏分收集。当一队的两名研究人员进行的，该协议需要八个小时的平均水平。 1。设立STA-PUT设备（图1） *** STA-PUT装置应放置在4℃的大冰箱或冷室，也可以容纳一个馏分收集器，如果是优选集合的该方法。 <ol><li>前一天晚上（或至少几个小时之前）?...

<ol>
	<li>Wistuba, J., Stukenborg, J., Luetjens, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Spermatogenesis.">Mammalian Spermatogenesis.</a> Funct. Dev. Embryol. 1, 99-117 (2007).</li><li>Hess, R. A., Cooke, P. S., Hofmann, M. C., Murphy, K. 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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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro,+short-term+culture+method+for+mammalian+haploid+round+spermatids+amenable+for+molecular+manipulation.">An in vitro, short-term culture method for mammalian haploid round spermatids amenable for molecular manipulation.</a> Mol. Reprod. Dev. 79, 19-30 (1002).</li></ol>

那些谁研究精子依靠动物模型的生精细胞样本，作为一种可靠的细胞培养系统尚未产生所有的生精细胞类型3存在。虽然生精细胞是从整个睾丸容易收集，只有混居的结果。这就带来了一个问题，对于那些谁希望研究这些细胞的特异性亚型，如减数分裂的细胞，圆形精子细胞和精子细胞凝结。三种不同的方法，目前用于分离睾丸生精细胞的亚型：STA-PUT，流式细胞仪，和淘洗6-13。后...

哺乳动物的精子发生是发生在几个阶段的睾丸1的曲细精管一个复杂的分化过程。简单地说，干细胞样的精原细胞驻留附近的曲细精管分化的上皮细胞和分化为精母细胞，然后进行减数分裂。减数分裂完成后，所得到的单倍体细胞，或圆精子细胞，精子发生，分化过程，包括细胞质和细胞核的压实的脱落。精子逐渐发展鞭毛，并进行延伸细胞核和冷凝，产生拉长，然后冷凝精子细胞，分别。最终的产品是精子，它被释放到精曲小管，并最终成为他们进一步成熟附睾管腔。 因为精子的过程依赖于特殊的激素和分子条件睾丸，可靠的体外培养体系为精子的整个过程中尚未被开发2,3。培养的方法已经被开发用于从干细胞产生“原始生殖细胞样细胞”和单倍体，“圆精子细胞样细胞”，但这些方法中还没有能产生大量的这些细胞并不能产生更高精细胞类型4,5。幸运的是，精细胞类型中尺寸显著不同，它允许从整个睾丸与液体梯度进行分离而获得的单细胞悬浮液。在STA-PUT方法，在这里表现出来，使用线性渐变的BSA和简单的沉淀根据大小和质量6-9分离睾丸生精细胞。 在STA-PUT方法具有比其他两种最广泛使用的方法几个优点分离生精细胞类型：流式细胞仪和淘洗10-13。在STA-PUT设备需要ONLŸ几件玻璃器皿专业组装在一个寒冷的房间或大冰箱。因而，它比用细胞分选器或淘析器更便宜。在STA-PUT方法产生更高的金额每个细胞类型和睾丸比可以通过流式细胞仪在可比时间段进行排序的细胞，虽然每个细胞群的纯度不高那些与流式细胞仪11获得。细胞分选，利用磁珠（磁性激活细胞分选，MACS）最近已成功地用于从一混合睾丸细胞群体富集的精原细胞，但它是目前不适于分离精母细胞或精子细胞由于缺乏适当的表面的知识的标记物14。过FACS或MACS的STA-PUT方法的一个附加的优点是隔离适于后续培养物的活细胞，因为，相对于最FACS协议，它不要求任何DNA或其他类型的染色能力。对于需要升研究生精细胞类型的ARGE收益率〜90％的纯度，在STA-PUT是一种理想的方法。

<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

哺乳动物的精子发生是发生在几个阶段的睾丸的曲细精管一个复杂的分化过程。目前，没有可靠的细胞培养系统，允许在体外分化精和精细胞的大多数生物学研究需要组织收获从象小鼠和大鼠的动物模型。因为睾丸含有大量的细胞类型 - 无论是无生精（睾丸间质，滋养，骨髓和上皮细胞）和生精（精原细胞，精母细胞，圆形精子细胞，精子细胞冷凝和精子） - 参与精子的生物学机制的研究需要隔离与这些不同类型的细胞的富集。在STA-PUT方法允许细胞的异质群体的分离 - 在这种情况下，从睾丸 - 通过一个线性BSA梯度。单个细胞类型具有不同的沉降速度根据CE滓LL大小，以及馏分富含不同类型的细胞可以被收集并在进一步的分析中使用。而STA-PUT方法不会导致细胞类型的高纯度的级分， 例如为可以与某些细胞分选方法获得，它不提供在每个级分高得多的产量总的细胞 （〜1×10 8个细胞/精细胞从7-8×10 8个细胞的起始种群类型）。这种高收益率的方法，只需要专门的玻璃器皿，可以在任何冷室或大型冷库中进行，使其成为具有有限的访问专门的设备，如荧光激活细胞分选（FACS）或淘析实验室的理想方法。

从STA-put过程的理想结果是细胞从睾丸根据细胞的大小和密度相当明显的分离。而细胞从睾丸中分离是通过BSA梯度沉淀，细胞的几个不同的频带可以观察到。细胞的任何团块往往会沉到梯度的底部，不会污染其他馏分。再往上升的梯度将是大体细胞和减数分裂的细胞。更远了梯度仍然会较小圆形精子细胞。在渐变的顶部将被浓缩精子细胞，精子和污染红细胞（这似乎是小圆细胞没有细胞核）。 <...

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

在STA-PUT方法允许对生精细胞的不同人群根据大小和密度的分离。

生精细胞类型使用STA把速度沉降分离

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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Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

分离的小鼠胚胎面外胚层和间充质

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...