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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 1.5 ml tube | Watson | 131-7155C | |
| 100 mm Petri dish | Corning, Falcon | 351029 | |
| 15 mL Centrifuge tube | Watson | 1332-015S | |
| 5 ml polystyrene tube with cell strainer snap cap (35 µm nylon mesh) | Corning, Falcon | 352235 | |
| 50 mL Centrifuge tube | Watson | 1342-050S | |
| 60 mm Petri dish | Corning, Falcon | 351007 | |
| 70 µm nylon mesh | Corning, Falcon | 352350 | |
| Cell sorter | Sony | SH800S | |
| Centrifuge | |||
| Collagenase, recombinant, Animal-derived-free | FUJIFILM Wako Pure Chemical Corporation | 036-23141 | |
| Collagenase, Type 1 | Worthington | LS004196 | |
| Cover glass | Fisher | 12-544-G | |
| Cytospin 3 | Shandon | ||
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Fisher | D1306 | working concentration: 0.1 μg/mL |
| Dnase I | Sigma | D5025-150KU | |
| Donkey serum | Sigma | S30-M | |
| Dulbecco’s phosphate-buffered saline (DPBS) | Gibco | 14190144 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11885076 | |
| Fetal bovine serum (FBS) | Gibco | 16000044 | |
| Hostone H1T antibody | gift from Mary Ann Handel | 1/2000 diluted | |
| Hank’s balanced salt solution (HBSS) | Gibco | 14175095 | |
| Hyaluronidase from bovine testes | Sigma | H3506-1G | |
| Phosphate buffered saline (PBS) | Sigma | P5493-4L | |
| Pipettemen | |||
| ProLong Gold Antifade Mountant | Fisher | P36930 | |
| rH2AX antibody | Millipore | 05-635 | working concentration: 2 μg/mL |
| Sperm Fertilization Protein 56 (Sp56) antibody | QED Bioscience | 55101 | working concentration: 0.5 μg/mL |
| Sterilized forceps and scissors | |||
| Superfrost /Plus Microscope Slides | Fisher | 12-550-15 | |
| SYCP3 antibody | Abcam | ab205846 | working concentration: 5 μg/mL |
| TWEEN 20 (Polysorbate 20) | Sigma | P9416 | |
| Vybrant DyeCycle Violet Stain (DCV) | Invitrogen | V35003 | |
| Water bath |
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