Name: isolation of murine spermatogenic cells using a violet-excited cell-permeable dna binding dye
Uploaded: 2021-01-14T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye at JoVE.com

我们感谢 Namekawa 、Yoshida 和 Maezawa 实验室的成员的帮助;凯蒂·格哈特编辑手稿;Mary Ann Handel 用于共享 H1T 抗体，辛辛那提儿童医院医疗中心 （CCHMC） 研究流细胞学核心，用于共享由 NIH S10OD023410 支持的 FACS 设备;科学研究资助（KAKENHI;17K07424）给T.N.;拉洛尔基金会博士后奖学金给美国;AMED-CREST （JP17gm1110005h0001） 到 S.Y.;阿扎布大学研究服务部资助研究项目， 教育、文化、体育、科学和技术部（MEXT）支持的私立大学研究品牌项目（2016-2019年）、研究活动启动资助计划（19K21196）、武田科学基金会（2019年）和Uhara纪念基金会研究奖励基金（2018年）对S.M;国家卫生研究院 R01 GM122776 至 S.H.N.

该协议遵循机构动物护理和使用委员会的准则（协议号）。IACUC2018-0040）在辛辛那提儿童医院医疗中心。
1. 用于制备睾丸悬浮液的设备和试剂设置
<ol><li>将每种酶储存在1x汉克斯的平衡盐溶液（HBSS）中，储存在-20°C（表1）。 注：在实验前随时准备。</li>
	<li>实验前一天：在4°C下用胎儿牛血清（FBS）收集管（1.5 mL管）。</li>
	<li>实验当天：将水浴设置为37°C。</...

<ol>
	<li>Griswold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis:+The+Commitment+to+Meiosis.">Spermatogenesis: The Commitment to Meiosis.</a> Physiological Reviews. 96 (1), 1-17 (2016).</li><li>Yoshida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Spermatogenesis+Reflects+the+Unity+and+Diversity+of+Tissue+Stem+Cell+Niche+Systems.">Mouse Spermatogenesis Reflects the Unity and Diversity of Tissue Stem Cell Niche Systems.</a> Cold Spring Harbor Perspectives in Biology. , 036186 (2020).</li><li>Rathke, C., Baarends, W. M., Awe, S., Renkawitz-Pohl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+dynamics+during+spermiogenesis.">Chromatin dynamics during spermiogenesis.</a> Biochimica et Biophysica Acta. 1839 (3), 155-168 (2014).</li><li>Maezawa, S., 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=SCML2+promotes+heterochromatin+organization+in+late+spermatogenesis.">SCML2 promotes heterochromatin organization in late spermatogenesis.</a> Journal of Cell Science. 131 (17), 217125 (2018).</li><li>Maezawa, S., Yukawa, M., Alavattam, K. G., Barski, A., Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+reorganization+of+open+chromatin+underlies+diverse+transcriptomes+during+spermatogenesis.">Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis.</a> Nucleic Acids Research. 46 (2), 593-608 (2018).</li><li>Bellve, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+culture,+and+fractionation+of+spermatogenic+cells.">Purification, culture, and fractionation of spermatogenic cells.</a> Methods in Enzymology. 225, 84-113 (1993).</li><li>Bryant, J. M., Meyer-Ficca, M. L., Dang, V. M., Berger, S. L., Meyer, R. G. <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+cell+types+using+STA-PUT+velocity+sedimentation.">Separation of spermatogenic cell types using STA-PUT velocity sedimentation.</a> Journal of Visualized Experiments. (80), e50648 (2013).</li><li>Gaysinskaya, V., Bortvin, A. <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+of+murine+spermatocytes.">Flow cytometry of murine spermatocytes.</a> Current Protocols in Cytometry. 72, (2015).</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 Part A. 65 (1), 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> Journal of Visualized Experimments. (50), e2602 (2011).</li><li>Gaysinskaya, V., Soh, I. Y., van der Heijden, G. W., Bortvin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+flow+cytometry+isolation+of+murine+spermatocytes.">Optimized flow cytometry isolation of murine spermatocytes.</a> Cytometry Part A. 85 (6), 556-565 (2014).</li><li>Romer, K. A., de Rooiji, D. G., Kojima, M. L., Page, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+mitotic+and+meiotic+germ+cells+from+male+mice+by+developmental+synchronization,+staging,+and+sorting.">Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting.</a> Developmental Biology. 443 (1), 19-34 (2018).</li><li>Lam, K. G., Brick, K., Cheng, G., Pratto, F., Camerini-Otero, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type-specific+genomics+reveals+histone+modification+dynamics+in+mammalian+meiosis.">Cell-type-specific genomics reveals histone modification dynamics in mammalian meiosis.</a> Nature Communications. 10 (1), 3821 (2019).</li><li>Geisinger, A., Rodriguez-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+the+Isolation+and+Characterization+of+Rodent+Meiocytes.">Flow Cytometry for the Isolation and Characterization of Rodent Meiocytes.</a> Methods in Molecular Biology. 1471, 217-230 (2017).</li><li>da Cruz, 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=Transcriptome+analysis+of+highly+purified+mouse+spermatogenic+cell+populations:+gene+expression+signatures+switch+from+meiotic-to+postmeiotic-related+processes+at+pachytene+stage.">Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage.</a> BMC Genomics. 17, 294 (2016).</li><li>Rodriguez-Casuriaga, 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=Rapid+preparation+of+rodent+testicular+cell+suspensions+and+spermatogenic+stages+purification+by+flow+cytometry+using+a+novel+blue-laser-excitable+vital+dye.">Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye.</a> MethodsX. 1, 239-243 (2014).</li><li>Trovero, M. F., 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=Revealing+stage-specific+expression+patterns+of+long+noncoding+RNAs+along+mouse+spermatogenesis.">Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis.</a> RNA Biology. 17 (3), 350-365 (2020).</li><li>Telford, W. G., Bradford, J., Godfrey, W., Robey, R. W., Bates, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+analysis+using+a+violet-excited+cell-permeable+DNA+binding+dye.">Side population analysis using a violet-excited cell-permeable DNA binding dye.</a> Stem Cells. 25 (4), 1029-1036 (2007).</li><li>Alavattam, K. G., Abe, H., Sakashita, A., Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+Spread+Analyses+of+Meiotic+Sex+Chromosome+Inactivation.">Chromosome Spread Analyses of Meiotic Sex Chromosome Inactivation.</a> Methods in Molecular Biology. 1861, 113-129 (2018).</li><li>Maezawa, S., 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=Super-enhancer+switching+drives+a+burst+in+gene+expression+at+the+mitosis-to-meiosis+transition.">Super-enhancer switching drives a burst in gene expression at the mitosis-to-meiosis transition.</a> Nature Structural &amp; Molecular Biology. , (2020).</li><li>Sakashita, 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=Endogenous+retroviruses+drive+species-specific+germline+transcriptomes+in+mammals.">Endogenous retroviruses drive species-specific germline transcriptomes in mammals.</a> Nature Structural &amp; Molecular Biology. , (2020).</li><li>Agrimson, K. S., 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=Characterizing+the+Spermatogonial+Response+to+Retinoic+Acid+During+the+Onset+of+Spermatogenesis+and+Following+Synchronization+in+the+Neonatal+Mouse+Testis.">Characterizing the Spermatogonial Response to Retinoic Acid During the Onset of Spermatogenesis and Following Synchronization in the Neonatal Mouse Testis.</a> Biology of Reproduction. 95 (4), 81 (2016).</li><li>Hogarth, C. 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=Turning+a+spermatogenic+wave+into+a+tsunami:+synchronizing+murine+spermatogenesis+using+WIN+18,446.">Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446.</a> Biology of Reproduction. 88 (2), 40 (2013).</li><li>Patel, 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=Dynamic+reorganization+of+the+genome+shapes+the+recombination+landscape+in+meiotic+prophase.">Dynamic reorganization of the genome shapes the recombination landscape in meiotic prophase.</a> Nature Structural &amp; Molecular Biology. 26 (3), 164-174 (2019).</li><li>Alavattam, K. 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=Attenuated+chromatin+compartmentalization+in+meiosis+and+its+maturation+in+sperm+development.">Attenuated chromatin compartmentalization in meiosis and its maturation in sperm development.</a> Nature Structural &amp; Molecular Biology. 26 (3), 175-184 (2019).</li><li>Adams, S. 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=RNF8+and+SCML2+cooperate+to+regulate+ubiquitination+and+H3K27+acetylation+for+escape+gene+activation+on+the+sex+chromosomes.">RNF8 and SCML2 cooperate to regulate ubiquitination and H3K27 acetylation for escape gene activation on the sex chromosomes.</a> PLoS Genetics. 14 (2), 1007233 (2018).</li><li>Wiltshire, T., Park, C., Caldwell, K. A., Handel, 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=Induced+premature+G2/M-phase+transition+in+pachytene+spermatocytes+includes+events+unique+to+meiosis.">Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis.</a> Developmental Biology. 169 (2), 557-567 (1995).</li></ol>

在这里，我们提出了一个实用和简单的协议，从单个成年雄性小鼠中分离精子细胞和精子的亚群。为了确保此协议的可重复性，有一些关键步骤需要注意。在酶消化之前，洗涤步骤旨在去除间分裂细胞;消化后，这一步有助于去除精子和碎片。洗/离心3次很重要。在我们的分离缓冲液配方中，几种不同酶的组合有助于将睾丸分离到单细胞悬浮液中，而不会造成过多的细胞损伤。轻轻移液以避免造成?...

精子生成是一个复杂的过程，其中一小群精子干细胞维持着大量精子在整个成年生中连续生产1，2。在精子生成过程中，动态染色质重塑发生在精子生成细胞经历美病产生单倍体精子3，4，5。分离美的精子细胞对于分子研究至关重要，并建立了几种不同的分离美亚精子细胞的方法，包括沉积分离6、7和荧光活性细胞分选（FACS）8、9、10、11、12、13、14、15、16、17。但是，这些方法有技术限制。虽然沉积基分离产生大量的细胞5，6，7，它是劳动密集型的。已建立的基于FACS的方法使用Hochst 33342（Ho342）根据DNA含量和光散射特性分离美的精子细胞，并要求FACS细胞分拣机配备紫外线（UV）激光8，9，10，11。基于FACS的替代方法要求转基因小鼠线表达荧光蛋白，精子生成12的同步，或细胞固定和抗体标签，不兼容分离活细胞13。虽然有另一种方法使用细胞渗透DNA结合染料，DyeCycle绿色染色14，15，16，17，此方法被推荐用于分离精子细胞从幼年睾丸。因此，迫切需要开发一种简单而坚固的分离方法，用于活体精子细胞，可应用于任何年龄的小鼠菌株，并可以使用任何 FACS 细胞分拣机进行。
在这里，我们描述这样一个长期寻求的细胞隔离协议使用染料循环紫罗兰（DCV）污渍。DCV是一种低细胞毒性，细胞渗透DNA结合染料结构类似于Ho342，但与激发光谱转向紫罗兰范围18。此外，与 DCG 相比，DCV 具有更广泛的发射频谱。因此，它可以通过紫外线和紫罗兰激光器，从而提高设备的灵活性，允许使用不配备UV激光的FACS细胞分拣机。此处介绍的 DCV 协议使用二维分离，具有 DCV 蓝色和 DCV 红色，模仿 Ho342 协议的优势。凭借这一优势，我们的DCV协议使我们能够从成人睾丸中分离出高度丰富的生殖细胞。我们提供详细的浇注方案，从一只小鼠的成年小鼠睾丸（从两个睾丸）分离出活的精子细胞。我们还描述了一个高效和快速的协议，用于准备单细胞悬浮从鼠标睾丸，可用于此细胞隔离。该程序需要很短的时间才能完成（准备单细胞悬浮 - 1小时，染色染色 - 30分钟，细胞分选 - 2-3小时：总计 - 4-5小时，视所需细胞数量不同; 图1）。细胞分离后，可以完成广泛的下游应用，包括RNA-seq、ATAC-seq、ChIP-seq和细胞培养。

<table>
	<tbody>
		<tr>
			<td>1.5 ml tube</td>
			<td>Watson</td>
			<td>131-7155C</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Corning, Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tube</td>
			<td>Watson</td>
			<td>1332-015S</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polystyrene tube with cell strainer snap cap (35 &#181;m nylon mesh)</td>
			<td>Corning, Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tube</td>
			<td>Watson</td>
			<td>1342-050S</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Corning, Falcon</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m nylon mesh</td>
			<td>Corning, Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>Sony</td>
			<td>SH800S</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, recombinant, Animal-derived-free</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>036-23141</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Fisher</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin 3</td>
			<td>Shandon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Fisher</td>
			<td>D1306</td>
			<td>working concentration: 0.1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma</td>
			<td>D5025-150KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>S30-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885076</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Hostone H1T antibody</td>
			<td>gift from Mary Ann Handel</td>
			<td></td>
			<td>1/2000 diluted</td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase from bovine testes</td>
			<td>Sigma</td>
			<td>H3506-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5493-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettemen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Fisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>rH2AX antibody</td>
			<td>Millipore</td>
			<td>05-635</td>
			<td>working concentration: 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sperm Fertilization Protein 56 (Sp56) antibody</td>
			<td>QED Bioscience</td>
			<td>55101</td>
			<td>working concentration: 0.5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sterilized forceps and scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost /Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>SYCP3 antibody</td>
			<td>Abcam</td>
			<td>ab205846</td>
			<td>working concentration: 5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>TWEEN 20 (Polysorbate 20)</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vybrant DyeCycle Violet Stain (DCV)</td>
			<td>Invitrogen</td>
			<td>V35003</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of murine spermatogenic cells using a violet-excited cell-permeable dna binding dye

分离美的精子细胞对于研究美因病和精子生成背后的分子机制至关重要。虽然有既定的细胞隔离协议使用 Hoechst 33342 染色结合荧光激活细胞分选，但它需要配备紫外线激光的细胞分拣机。在这里，我们描述一个细胞隔离协议使用染料循环紫罗兰（DCV）污渍，低细胞毒性DNA结合染料结构类似于霍赫斯特33342。DCV 可以同时受到紫外线和紫罗兰激光器的兴奋，从而提高了设备选择的灵活性，包括未配备紫外线激光的电池分拣机。使用该协议，可以分离出三个活细胞亚群在美的正头 I，包括麻黄素/酶，帕奇滕，和二苯甲酸精子细胞，以及后美的圆形精子。我们还描述了一个协议，用于准备从鼠标睾丸的单细胞悬浮。总体而言，该过程需要很短的时间才能完成（4-5 小时取决于所需的单元数量），这有利于许多下游应用。

此排序协议的代表性结果如图3 所示。两个睾丸（一只鼠标）的总分拣时间通常为3小时左右，这取决于细胞悬浮液的浓度和分拣速度。分类后，通过SYCP3和+2AX的免疫处理确认精子细胞的纯度（图3A）。排序L/Z、P、D精子细胞分数的代表性纯度分别约为80.4%、90.6%和87.6%（图3C）。我们根据先前19日公布的标准确定子阶段</...

Watch this Scientific Journal Video about Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye at JoVE.com

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

在这里，我们提出了一个简单而有效的方法来从成年小鼠睾丸中分离活的美病细胞和后美生菌细胞。使用低细胞毒性、紫罗兰兴奋的DNA结合染料和荧光激活细胞分选，可以分离高度丰富的精子致原细胞群，用于许多下游应用。

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

这种新的有效方法，隔离亚群的精子细胞和精子从成年小鼠可用于研究微生和精子发生背后的分子机制。此方法使用低细胞毒性DNA结合染料与广泛的激发光谱，可应用于大多数目前使用的FACS订单。此方法非常简单。最具挑战性的方面是流式细胞仪门控，但详细的门控策略应有助于使协议适应其他细胞分拣机。演示这个程序的将是来自佐藤名川实验室的研究助理叶玉汉。从一只八周大的雄性小鼠中采集两个睾丸后，将组织放在含有两毫升冰冷PBS的60毫米培养皿中，取出图尼卡藻类。用钳子轻轻分离睾丸，以稍微分散半尼的管。将小管转移到100毫米培养皿中一滴新鲜的冰冷PBS中。使用钳子轻轻解开管状液，并在三个额外的 PBS 液滴中清洗管状液，正如刚才演示的。最后一次洗涤后，将未解开的半尼弗管放入15毫升的管子中，内含两毫升分离缓冲液，在37摄氏度下孵育20分钟。在孵育结束时，使用1000微升移液器轻轻移液管20次，然后将管子返回孵化器6分钟。在孵化结束时，移液组织再移液20次，然后将管子返回孵化器。三分钟后，轻轻移液10次，或直到没有可见的组织片段停留，然后停止与10毫升的FACS缓冲液分离。然后，将离心机离心组织悬浮两次，使用额外的10毫升，用于新的FACS缓冲液进行第二次洗涤，以去除精子和尽可能多的碎屑。要染色细胞进行流动细胞测量分析，请将颗粒重新悬浮在三毫升的 FACS 缓冲液中。并通过70微米尼龙细胞过滤器过滤电池悬浮液，过滤成50毫升的管子。计数后，将10%的细胞转移到冰上的新管中，作为未污染的阴性对照。将六微升的 DCV 污渍彻底混合到剩余的细胞悬浮液中。在黑暗中37摄氏度下孵育30分钟后，每10分钟轻轻摇晃一次，在细胞中加入5微升DNase I。并通过35微米尼龙网过滤器过滤细胞，放入冰上5毫升的FACS管中。对于细胞的流动细胞测量分析，在流分析软件中创建新实验，在工作表工具菜单下，单击新密度以创建线性比例上的向前散射区与反散射区密度图。单击新的直方图，在对数刻度上创建 DCV 蓝色直方图图。然后单击"开始"和"记录"开始处理未污染的样本。在样品运行时，单击探测器和阈值设置以调整正向和侧面散射 PMT 电压，将未污染的电池放在前进和背面散点图的刻度上。调整 jappy 通道的 PMT 电压，以定位 DCV 蓝色直方图对数图第一个十年中 DCV 负位的位置。然后，单击"停止"以卸载未污染的示例。短暂涡旋并加载染色样品后，单击下一管为样品创建新工作表。将细胞计设置为至少获取六个事件的 10 倍，然后单击"开始"和"记录"。接下来，单击新密度以创建向前散射高度与向前散点宽度。和直流V蓝色与DCV红色密度图在线性比例。在前进散射区与反散射区域密度图上，使用多边形工具绘制一个名为"单元格"的栅极，以包括大部分单元格并排除小碎片。将此栅极应用于正向散点高度与正向散点宽度图，并使用矩形工具绘制单个单元格的栅极以排除非单个单元格。将单个单元的栅极应用于 DCV 蓝色与 DCV 红色密度图，并调整刻度以捕获扩展轮廓。使用多边形工具绘制 DCV 门以排除未污染的单元格和侧总体，并应用该门以线性比例将门应用于 DCV 蓝色直方图图。三个主要峰是指不同的1C、2C和4CDNA含量。在线性比例上创建第二个 DCV 蓝色与 DCV 红色密度图。后门DCV门到地块，以找到1C和4C人口。在线性比例上创建另一个 DCV 蓝色与 DCV 红色密度图。并使用椭圆工具在 1C 总体上绘制门。使用多边形工具用连续曲线对 4C 总体进行门控。并在线性比例上创建另一个 DCV 蓝色与 DCV 红色密度图。使用多边形工具创建一个 4C 一个门。在一个新的向前侧散射图中，使用椭圆工具创建帕奇滕、二氯丁二烯和麻黄素、zygotene精细胞门。绘制所有门后，以线性比例创建新的 DCV 蓝色与 DCV 红色点图，以应用 4C 门，以确保三个填充在门内以连续顺序排列。接下来，在线性比例上创建新的向前散射区与反散射区密度图，并选择细胞的统一大小作为纯圆形精子组。分类后，分离的钩端素、酶氨酸、帕奇泰尼和二甲苯精细胞分馏的代表性纯度通常介于SYCP3和伽玛H2AX免疫处理确认的80%至90%之间。圆形精子的纯度可以通过与DCV结合SP56和H1t染色的细胞核染色来确认，并且通常也是90%左右，分离细胞群的生存能力通常超过95%，单个成年小鼠中每个分数的平均总产量通常足以进行各种下游分析。排序的细胞可用于各种下一代测序分析，以更好地探索精子生成过程中的基因表达和调控。

Research

JoVE Journal

Developmental Biology

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

使用小鸡GFP 神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

科研

发育生物学

Murine Dermal Fibroblast Isolation by FACS

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...