The authors would like to acknowledge the NHMRC for career development award and grant support APP1066336 which supported this work.

1.准备文化传媒和菜名<ol><li>使200毫升Biggers的惠顿和惠顿（BWW）17工作溶液中加入5.54克氯化钠，0.356克氯化钾0.25克CaCl 2，0.162克的H 2 KO 4 P和0.294克硫酸镁到1升的Milli-Q水。这是原液并可以保持在4℃最多一个月。 </li><li>从该原液中，添加碳酸氢钠的420毫克- ，200毫克葡萄糖，6毫克丙酮酸钠，600毫克牛血清白蛋白，0.74毫升乳酸钠和4.0毫升HEPES缓冲剂至193毫升BWW股票。这是工作溶液，并在使用前一直制作新鲜的当天和平衡至37℃。 </li><li>使插管，取聚乙烯（PE）管具有0.4mm的内径和1.1毫米的外径和保持在一个较低的热（通常为甲醇火焰）使得所述管开始熔化。立即拉管线outwARD伸展，使外径窄。 </li><li>切割以产生一个端其允许输精管容易插管变窄。 </li><li>切插管的另一端至约15公分。插入地下30针插入钝端，并附加3毫升注射器将其（完全缩回）。 </li><li>通过削减PE管（4.2毫米的直径，6.4 mm外径）的20厘米长做吸喉舌。插入一个喉舌成一端。 </li><li>插入微毛细管玻璃管保持器和玻璃微毛细管本身（一般为3微升为小鼠，40微升为一大鼠）。 </li></ol>从鼠标2.去除附睾<ol><li>根据具体到每个机构IACUC批准的安乐死程序老鼠。 </li><li>取的动物，使一个小切口在阴囊以暴露附睾。拉睾丸和附睾出使用一对钟表＃5镊子的空腔。 </li><li>切输精管，使得至少1-2厘米保持连接到马尾附睾。此外，切断近端输出小管和组织连接到附睾睾丸和删除整个男性生殖轨道。 </li><li>将解剖显微镜的放大倍数范围5-40X的秩序下，整个男性生殖轨道。 </li></ol> 3. Cannulating附睾<ol><li>胶带向下插管到显微镜，使得1-2厘米的变窄（圆锥形）的端部是自由和可见光通过透镜。 </li><li>采取一对钟表＃5镊子轻轻扣着输精管每一侧并拔出输精管到套管，使得所述套管进入输精管。 </li><li>采取非吸收性的黑色编织丝绸处理（尺寸5-0）的长度和领带绕空心输精管结。确保结紧紧地按住输精管内插管时，气压FR嗡注射器被应用。 </li></ol> 4.逆行或骶管附睾精子的反冲<ol><li>使用制表大师＃5镊子，抢马尾附睾的远端，并删除白膜，以公开一个附睾小管。 </li><li>使用镊子，轻轻一拉小管暴露出来，然后逗开以创造一个开放的精子被释放。 </li><li>轻轻地推上3毫升（小鼠）或20毫升（鼠）注射器，以便排出空气从注射器进输精管。在适当的压力下，精子将开始慢慢出来破碎条状细管的马尾附睾的前端。此时，施加吸力到吸嘴吸取精子放入玻璃毛细管。注意：通常在小鼠，2-3微升精子可以根据动物的年龄而获得。一只老鼠的意志，平均产量30-40微升。 </li></ol> 5.洗涤和裂解的精子为蛋白质组学准备<ol><li>通过轻轻地吹对着话筒或附加一个注射器和排出空气，以便将精子推回出毛细管的排出从玻璃毛细管精子进入BWW溶液1 ml溶液（37℃）。 </li><li>一旦精子细胞扩散，洗3次（300×g离心，5分钟）用1ml BWW以除去污染性蛋白。最后一次洗涤后，除去上清液。注意：在这一点上，精子细胞可以冷冻供以后使用。 </li><li>增溶用4％CHAPS，2M硫脲，和50mM的Tris，pH 7.4中的蛋白质1小时，间歇漩涡。 </li><li>裂解细胞，离心（10,000×g离心，20分钟）后，取上清液并转移到新的管中。注意：在这一点上，蛋白质定量可以执行。 </li></ol> 6.二硫键还原和烷基化<ol><li>添加10mM的DTT作为最终浓度以裂解蛋白质，vort前和在室温下孵育30分钟。 </li><li>添加50毫idodoacetamide作为最终浓度以裂解物，涡旋并在室温下孵育在黑暗中30分钟。 </li></ol> 7.降水<ol><li>甲醇氯仿沉淀样品中加入1体积裂解物（400微升作为一个例子），加入甲醇1体积（400微升）和0.5体积（200微升）氯仿。 </li><li>涡旋样品和自旋（10,000 xg离心，2分钟）。 </li><li>需要注意的是两个阶段会出现以下离心。丢弃顶层（注意不要打扰接口）的所有，但为2 mm。 </li><li>加入1倍体积的甲醇中（400微升），转化管轻轻1-2x混合并再次旋转（10,000×g离心，15分钟）。弃上清浪费和空气干燥颗粒为3-4分钟。 </li></ol> 8.胰酶消化<ol><li>重组胰蛋白酶在25mM碳酸氢铵含1M尿素的50：1的比例（w / w;蛋白：胰蛋白酶）并孵育OVErnight在37℃下优选在700rpm在一个恒温。 </li><li>自旋（10,000×g离心，15分钟）以沉淀未消化的材料。转移上清液至新管中。 </li></ol> 9.磷酸化肽富集<ol><li>从胰蛋白酶进行纯化和磷酸富集消化如前所述18。稀释的胰蛋白酶肽10倍于DHB缓冲液[DHB缓冲器组成：350毫克/毫升2,5- dihydrobenzesulfonic苯甲酸（DHB），80％（体积/体积）ACN（乙腈），2％（V / V）TFA（三氟酸）]，并适用于干燥二氧化钛珠（200微克）。 </li><li>留在旋转器上在室温下搅拌1小时。 </li><li>洗DHB缓冲区样本，旋转和上清。然后洗样品三次，洗涤缓冲液[80％ACN（V / V），2％TFA（V / V）]，除去DHB。 </li><li>在最后的旋转后，直接加入25微升2.5％的氢氧化铵，pH值≥10.5溶液洗脱使用洗脱缓冲液的磷酸。 S针，去除上清。立即用中和0.3〜微升甲酸。 </li></ol>

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The authors declare that they have no competing financial interests.

对于精子的一个成功的和可重复的蛋白质组分析的关键步骤是：1）纯度的原料; 2）去除多余的盐和洗涤剂; 3）变性蛋白，以它们的全部范围，以使胰蛋白酶消化高收率蛋白和4）最小化的处理，以减少肽的损失样品。 为了成功地反冲马尾附睾，有必要确定从精子将退出区域。在大鼠和小鼠中的情况下，这是在所述凹区域的顶点，在附睾的尾部区域的中间（参见图2A）。如果有进一步的来向输精管，反冲更容易，成功率一般较高。然而，这是以精子数的损失。可替代地，如果一个人试图移动至更近侧到语料库，那么压力的量所需的精子推回通过epididym人管往往是如此之高，是破坏附睾不可避免地发生。 传统上执行使用水饱和的矿物油和作为介质在注射器本身平衡盐溶液以除去精子22-25附睾的反冲。这两个程序都是潜在的问题LC-MS的。首先，矿物很可能会阻止必须采取这样没有结转过程中的纳米C18纳米列基本上都是采用的全球蛋白质组学分析和护理。如果发生这种情况，就不可能继续并将样品实质上丢失。这可以通过使用BWW或在注射器的其他平衡盐溶液，然而来克服，这虽然是成功的，我们很快认识到，许多所述精子成为能动只要BWW溶液开始接触，并与尾附睾混合细胞。为了解决这个问题，我们简单地反冲与空气的精子。不仅是资格赛精子两者均媲美的基于液体的方法，但该数量是相同的。 磷酸化蛋白质组学也许是确定哪些信号通路是发生在精子后射精的唯一途径之一。一个我们正在调查的主要途径是获能的过程。精子必须经过“获能”才能够结合到一个鸡蛋。在实践中，这是通过孵育精子一段时间基本实现（;大鼠1.5小时;小鼠40分钟人类3-24小时）与血清白蛋白BWW溶液。以前，我们和其他人已经表明对参与获能几个激酶的作用。兴趣的，缺失的PKA的μII产生的小鼠的精子游动自发体外 ，但不能经受过度活化26。后者是获能的标志，由此精子改变其泳图案从高速，低幅度，以一低速度，高振幅摆动频率。我们已经表明参与这一过程的下游激酶包括PP60-中国证监会（SRC）13,27 C-是28和C-ABL 14。有趣的是，抑制SRC的停止获能依赖的酪氨酸磷酸化13。然而，这可以用冈田酸来克服，这表明SRC不直接参与在一般性发病酪氨酸磷酸29的，但可以调节磷酸酶29。使用BWW作为介质，迫使精子出附睾的问题是，一旦被激活，小鼠精子只需要大约40分钟，以与能力。鉴于精子隔离可能需要5分钟/小鼠和常数小鼠在实验中使用，那么精子将到期的在实验开始时的不同阶段。为了克服这个，空气压力可用于从尾附睾精子推入玻璃套管。不仅所有的精子INACT香港专业教育学院和基本上如它们将在尾环境中找到，它使得有可能比较非能动和能动磷酸化蛋白质组学。 样品处理对于磷酸化蛋白质组学应在两个不同的功能状态进行比较的精子时被保持在最低限度。以上蛋白质沉淀1）的其他传统方法使用甲醇氯仿降低需要额外的洗涤步骤，2）去除盐和脂肪的几乎所有痕量和3）具有经过验证的能力超过TCA 19以沉淀低丰度的蛋白质。前胰蛋白酶消化的蛋白质沉淀，建议因为这不仅有助于变性蛋白（其有助于胰蛋白酶消化），但除去许多在MS-不相容代谢产物存在于细胞内的。 精子磷酸的比较可以以多种方式来完成。在基层，所识别的磷酸肽的一个样本的简单比较，以在另一在被称为“谱计数”的过程中采样可以做到的。但是批评一直处于这种方法主要是因为早期的蛋白质组学研究用低重复（对于更详细的讨论参见Lundren 等 30）。一个更复杂的方法是看该肽母体质量的强度，并与其它样品（无标记的比较）进行比较。在图4所示的例子中，肽不存在于来自非能动（ 图4上部），但目前在游动（ 图4下）精子从M / Z 650-670可以看出。这个过程中，被称为基于MS的标签免费量化是一个无标记的战略。 常用来降低所需蛋白质的定量运行的量的另一种策略是使用同位素。作为同位素的质量是不同的，质谱仪可以用来比较ELUT的强度荷兰国际集团肽。然而，与大多数其它细胞类型，某些同位素标记不能适用于精子。例如，通过添加稳定同位素（1重同位素被添加到一个样品，而较轻的版本被添加到另一个）可在组织培养物中使用。当同位素得到并入蛋白，蛋白组学分析可以通过组合两个样品（复用）进行。然而，在精子的情况下，这不能做，仅仅凭借的一个事实，即1）同位素标记需要几个通道（最多8个）在组织培养和2）的精子细胞不具有核基因的转录和翻译，因此，他们不能纳入同位素其所有的蛋白质而已。解决这个的一种方式是订购放射性标记的小鼠，但是，这些是众所周知的昂贵。另一种方法是使用一种化学标记。当非获能小鼠用获能的小鼠相比，这已经完成。在这种情况下，A D 0以及D <sub&gt; 3 -label用一个样品和另一个在质谱仪31之间进行区分。其他方法可以包括使用的iTRAQ（等压标记相对和绝对定量），由此赖氨酸被化学不同质量同位素标记的; ICAT（同位素编码的亲和标签），由此半胱氨酸标记有不同质量的同位素和重链和轻水标记。 在每一个情况，但是，有一点必须牢记。该MS仅报告什么是存在于样品中，而这是发生在该样品到这一点，一切的反映。最小化的样品处理，同时最大限度地提高产量在每一步是必要的一个成功的蛋白质组学分析。

从睾丸已经出现，精子是高度分化又显着，这些细胞是未成熟1,2。因此，它们缺乏完整的功能，包括游泳的能力，并结合到卵母细胞3。虽然精子细胞的进一步成熟是必需的，这无法通过典型途径发生，因为在此阶段的细胞分化的过程中，它们是不能的基因转录和进一步蛋白质生物合成4。生物的能力是赋予精子，因为它们离开睾丸和进入被称为附睾5男性生殖系统的一部分。附睾是一个紧密盘绕，高度分化的管连接所述输出小管（睾丸）输精管和存在于所有雄性哺乳动物6。精子在附睾血统逐步收购其受精能力，依靠不断变化的环境，鲁米那是CR由本地附睾上皮细胞7 eated。存在于附睾环境行为的各种分泌和再吸收的活动来修改精子本身，改变它的蛋白质，碳水化合物和脂质组合物6。附睾的重要性，尤其是最初的“人均”这个结构的地区已经采用手术结扎技术8被例证。精子通过传出神经导管结扎睾丸内保留完全缺乏任何身份受精的卵母细胞9-11。 除了附睾环境内的精子功信号转导通路对翻译后修改现有的精子细胞的补充。例如，从附睾的早期区域精子显示不同的蛋白质磷酸化模式相比，这些后者的区域5,12。这并不奇怪，因为在C巨大差异apability来自这些地区的精子。例如，精子从早期，附睾头都immotilite。与此相反，精子细胞从马尾区域将经受向前一旦放置在等渗介质渐进运动。由于附睾精子细胞是不能脱从头蛋白质生物合成，所有的固有途径调节精子功能必须通过翻译后修饰（PTM）。因此，按理说，蛋白质组学，特别是翻译后修饰，如磷酸化的调查必须是一个主要的推力，如果我们要了解精子细胞成熟。 另一种方法以从epididymidies获得精子使用复古反冲13-16。虽然这种技术是肯定比较费时，由操作者需要更大程度的技术人员，对精子获得一致表明纯度超过99.99％。此外，不同于所有其它技术，精子CAN为分离处于静止状态，使得能够研究如何精子活力被启动。样品制备是用于蛋白组学分析中最重要的方面，精子隔离已成为精子蛋白质组学研究中最重要的方面之一。该协议提供了有关如何从精子马尾附睾被隔离的解释。在此之后，在TiO 2磷酸富集过程概述，关于如何从高度分化的精子细胞中提取的肽的具体参考。该MS的方法可以用来区分改变磷酸如果一个人尾附睾精子在一种状态比较（immotile或非获能）到另一个（能动，获能，顶体反应等）使之成为一个有效的方法来研究的精子功能。

<table border="0" cellpadding="0" cellspacing="0" width="687">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,5-dihydroxy-1,4-benzoquinone</td>
			<td>Aldrich</td>
			<td>195464</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-D Quantitation Kit</td>
			<td>VWR (GE Healthcare)</td>
			<td>80-6483-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonia Solution 25%</td>
			<td>Merck</td>
			<td>1.05432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Bicarbonate</td>
			<td>Sigma</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate</td>
			<td>Research Organics</td>
			<td>1300C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>BDH</td>
			<td>10077.6B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">di-sodium hydrogen phosphate</td>
			<td>Merck</td>
			<td>1.06580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formic Acid</td>
			<td>Sigma</td>
			<td>O6440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamine-L</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes (free acid)</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodoacetic acid free acid</td>
			<td>Sigma</td>
			<td>I4386</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactic Acid</td>
			<td>Sigma</td>
			<td>L1750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride</td>
			<td>Res. Org.</td>
			<td>0090M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Merck</td>
			<td>10158.6B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll (sterile)</td>
			<td>VWR (GE Healthcare)</td>
			<td>GEHE17-0891-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphatase Inhibitor Cocktail Halt</td>
			<td>ThermoFisher Scientific (Pierce)</td>
			<td>PIE78420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Hydrogen Carbonate</td>
			<td>Medical Store</td>
			<td>CH 600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S3817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride&#160;</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium di-hydrogen phosphate</td>
			<td>Merck</td>
			<td>1.06346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrogen carbonate</td>
			<td>BDH</td>
			<td>10247.4V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Ultra Pure Grade</td>
			<td>Amresco (Astral)</td>
			<td>AM0497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin Gold-Mass Spec Grade</td>
			<td>Promega</td>
			<td>V5280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td>Sigma</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zinc Chloride</td>
			<td>Sigma</td>
			<td>Z0173</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4 mm polyethylene tubing</td>
			<td style="width:175px;">Goodfellow</td>
			<td style="width:129px;">ET317100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30 gauge needle</td>
			<td style="width:175px;">Terumo</td>
			<td style="width:129px;">NN2038R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 mL syringe</td>
			<td style="width:175px;">Terumo</td>
			<td style="width:129px;">SS035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4.2 mm polyethylene tubing</td>
			<td style="width:175px;">Goodfellow</td>
			<td style="width:129px;">ET317640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Braided silk (5-0)</td>
			<td style="width:175px;">Dynek</td>
			<td style="width:129px;">CSS0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid</td>
			<td>Sigma</td>
			<td>299537</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TiO2 beads (from disassembled column)</td>
			<td style="width:175px;">Titansphere</td>
			<td style="width:129px;">6HT68003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eppendorf Tube</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:129px;">22431081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C7B30</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:129px;">C0856</td>
			<td></td>
		</tr>
	</tbody>
</table>

phosphopeptide analysis of rodent epididymal spermatozoa

精子是相当独特的之间的细胞类型。虽然产生于睾丸，既核基因的转录和翻译被关闭一次预光标圆形细胞开始伸长和分化成什么形态识别为精子。然而，精子是很不成熟，没有能力的蠕动或卵子的认可。这两个事件的发生，一旦精子中转称为附睾一二级机构。在〜12天通道，它需要一个精子细胞穿过附睾，现有蛋白质的翻译后修饰发挥细胞的成熟中发挥关键作用。这样的一个主要方面是蛋白质的磷酸化。为了表征磷酸化事件中精子成熟发生，无论纯的精子细胞群和预分馏磷酸的必须建立。使用反冲技术，一种用于纯精子Ò隔离从远端或尾部附睾F高品质和产量概述。的步骤为溶解，消化和预分馏通过TiO 2的亲和层析精子磷酸肽进行说明。一旦分离，磷酸可注入的MS中的精子的成熟过程，以确定在特定的氨基酸残基都蛋白质磷酸化事件和量化的磷酸发生的水平。

从任何蛋白质组学分析的结果的质量在很大程度上依赖于起始原料。随着现代MS，在样品制备轻微污染很容易回升。因此，重要的是，在精细胞蛋白质组学的情况下，选择一种方法，使高纯度的精子。如示于图1中 ，逆行反吹用于从马尾附睾检索精子细胞。这涉及cannulating输精管用PE管材，它允许一个缓慢申请气压从连接注射器。 图1A展示马尾附睾与输精管。 图1B是密切如何输精管绑起来拍哪里插管被插入。在这样做的，因此，精子释放从尾附睾1细管的端部切下。 如图2A所示 ，精子从caud的顶点区域提取一个附睾和被吸入到处于静止状态（ 图2B）的玻璃毛细管。这具有比传统的“游式”的方法，而不是其中的是前和运动的激活后执行精子的phoshoproteomic分析的能力的几个优点。精子可以被驱逐到自己选择的媒体上。 图2C（左侧）演示了如何精子驱逐到BWW媒体后，立即寻找。许多细胞被成群在一起。然而，该细胞的10分钟后的能力证明，因为它们游出至均一到溶液中（ 图2C，右手边的细胞）。由于反冲技术对精子细胞的影响非常小，我们得到接近100％的蠕动，将细胞分散迅速进入媒体没有外部挑衅。在一个典型的反冲实验，精子恢复从瑞士包含鼠标1-5之间×10 6个细胞。这是高度依赖于动物的年龄，并且可以从小鼠的不同菌株而异。在挪威鼠，我们通常得200×10 6个精子，±20％。 图2D表示精子从大鼠马尾附睾中得到的纯度。 除了样品本身，关于试样制备的主要问题之一是胰蛋白酶消化的产率。蛋白质沉淀起着重要的作用，不仅在去除不需要的洗涤剂和盐，这两者都是与MS不相容，而且在变性蛋白质。我们已经发现甲醇 - 氯仿沉淀效果最好。不仅这一程序被报道以沉淀低丰度蛋白比其它包括TCA沉淀19更好，但它有另外的优点。以下TCA沉淀，通常有必要中止TCA沉淀的可保持相当后，调整pH值酸性。虽然三氯乙酸沉淀的沉淀可洗在高的有机溶液，以除去酸，这增加了样品处理和结果的不可再现。失败，以中和酸会导致不良的胰蛋白酶肽的产率。甲醇-氯仿沉淀，不仅快速，但不会酸化的样品。 图3示出了蛋白质沉淀典型地从100微克的下部和上部间期间样品的观察。 磷酸肽的分离可发生在许多方面;然而重复性取决于该样本已处理直至并包括这个阶段。一种更常见的，显影方法是二氧化钛，最初是由该组马丁拉森18,20,21的开发。虽然被描述为一个过程，微柱的使用，我们可以使用批处理色谱获得可再现的数据。该进程的成功在很大程度上依赖于洗脱缓冲，这一定是疯了<html> Ë用正确的成分;否则磷酸不洗脱的。 协议和有代表性的数据从TiO 2的再现性富集从马尾附睾精子磷酸在非能动（ 图4上）和能动（ 图4下）从米状态/ Z 650-670可以看出。我们已经证明，这是一个使用生物学重复17即使极可重现的技术。蓝色条纹出现随着时间的推移是肽从C18纳米柱作为乙腈的浓度增加洗脱。显然，在能动人群（n = 3中所示，每组8典型地运行），有一个肽簇，与周围的651.5沓范围内的单一同位素质量是在非能动范围完全不存在。串联质谱然后用于鉴定该肽。 546 / 51546fig1highres.jpg“/&gt; 图1：马尾附睾插管（A）图片马尾附睾。插管本身被录音下来以暴露预拉端的大约1-2厘米。这是用细＃5watchmakers钳插入输精管。（B）的套管到位绑细丝线绕含输精管和套管两种段举行。从小鼠，约2-3微升尾椎附睾细胞和流体被收集在1×10 6个/μl的浓度。从大鼠（示出），大约30-40微升的流体都在相似的浓度获得的。 <img alt="图2" src="/files/ftp_upload/51546/51546fig2highres.jpg" /> 图2：用于收集主管附睾精子玻璃毛细管。 （A）从马尾e的顶点一小管<html> pididymis是孤立和破碎。近似位置从这种情况被示出。（B）中的图像的顶部示出了一个空的玻璃毛细管和底部示出了一个从先前反冲老鼠。不存在血液污染和最小的上皮细胞污染。（C）由于精子仍然未稀释的附睾液，他们还没有开始启动。当精子最初排出到BWW溶液，他们出来作为一个紧凑“串”（左手侧）。细胞的能力是很容易确定的，因为在10分钟后大部分精子游进而没有任何外部干扰（右手侧）溶液中。（D）的尾部附睾细胞的等分试样的图像，他们已经离开后，立即向游出表明该细胞的纯度。 g3highres.jpg“/&gt; 图3：甲醇-氯仿沉淀一旦精子已溶解时，可溶组分是甲醇-氯仿沉淀，以确保蛋白质和去除污染物质的变性。蛋白质沉淀出现的甲醇/水和氯仿相之间的界面上。顶层被小心去除，以便不打扰此粒料。是很常见的离开大约2-3毫米的上层相的，以便无蛋白质丢失。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51546/51546fig4highres.jpg" width="600" /> 图4：典型的TiO 2富集的磷酸信息使用所描述的协议中，尾附睾精子中分离在任一immotile（顶部，N = 3）或能动状态（底部，N = 3）。随着〜651.5 D中单同位素质量肽集群一个被示出为存在于能动种群但完全不存在在immotile那些（箭头）。这种性质的比较是被称为“无标记（基于MS）”。肽质量和保留时间，然后用于靶向用于串联质谱的化合物，并确定从其中衍生的肽的蛋白质。

Watch this Scientific Journal Video about Phosphopeptide Analysis of Rodent Epididymal Spermatozoa at JoVE.com

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

phosphopeptide-analysis-of-rodent-epididymal-spermatozoa

Research

JoVE Journal

Biology

12.8K 次观看.  University of Newcastle. Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

视频: Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

科研

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

鼠类外科的原则，为新的外科医生

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

转基因检测鼠类量化男性生殖细胞突变频率

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

啮齿动物心脏中的病毒转基因表达和心律失常风险的评估

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...