我们感谢Kymberlee奥布莱恩，西莉亚·摩尔和爱德华Tronick（心理学马萨诸塞州，波士顿大学系）提供本研究分析了人的头发样本，以及斯蒂芬芬兰语和Amanda DETTMER（比较行为学，NICHD的实验室）进行提供恒河猴头发样本。最初的开发和持续利用这种方法已经得到了美国国立卫生研究院RR11122为MAN

1。样品采集和存储<ol><li>人发<ol><li>固定头发进行采样（最高铅笔宽度，直径），用橡皮带或夹子的整个长度。剪下头发紧贴头皮的可能（注意不要刻痕的皮肤），用干净的剪刀。注:标准取样面积是颅骨的后部顶点。 </li><li>一些研究者已经报道在人的头发CORT水平的下降与从头皮6，这可能是由于反复暴露于水和香波7被洗出的距离（虽然看Manenschijn 等人 8和Thomson 等人在9为相反的结果没有下降段观察），其中为了尽量减少这种潜在的"冲洗效应"的影响，我们建议近端收集头发段的长度不超过3厘米（假设为1厘米/ 10个月的平均增长速度，更大的头皮3厘米段包包含了C口服补液疗法，已沉积在过去大约3个月）。要做到这一点，用一把尺子来衡量3厘米从第一刀，切再次产生3厘米的样品。注意:一旦头发被切成需要的长度，它不再是必要的，以保持链对齐，除非该研究涉及切割样品放入单独的长1厘米段，建立CORT沉积回顾展在日历的时间段之前的采样6。 </li><li>将头发样品在由铝箔制成，干净的纸信封，或用于样品洗涤的类型的15毫升螺旋盖聚丙烯离心管中的一个小袋。作为CORT是在毛发2极其稳定，样品可无限期地贮存于-20℃，如果需要，在环境温度下运送过夜。 </li><li>毛发收集过程应实行志愿者和实践样本应着手全面研究之前进行权衡。注意:头发样本为SMALL为5-10毫克可以使用此处描述的方法进行分析，虽然理想的是收集样品&gt; 10毫克，以减少获得EIA读数低于最低CORT标准的可能性。 </li></ol></li><li>猴毛<ol><li>从颈部的使用标准的动物剪颈背的头发剃光（100-250毫克）。刮胡子尽可能靠近皮肤​​越好，小心不要刻痕的皮肤，引起出血，因为相比那些在头发发现血液中皮质酮水平是非常高的。注:颈部被选为常规取样，因为它一般是毛发的良好来源，可以为头发再生之前，重采样很容易观察到。 </li><li>将头发样品在由铝箔或15毫升螺旋盖聚丙烯离心管中的一个小袋。猴头发样品应当存放和运输上述用于人的头发样品的相同方式。 </li><li>由于猴毛，不像人类头发，长到一定长度，然后STOPS生长11，它通常是不可行的在这种情况下，使用从皮肤作为日历CORT沉积距离。然而，如果动物可以被采样一次以上（例如，在定期例行健康检查），理想的是执行初始剃须设置一个基准时间点，然后reshave同一区域后所需的时间段已经过去。 CORT所述第二样品中的剃须-reshave间隔，这允许样品的CORT含量HPA活性超过该区间5的精确归因于沉积。 </li></ol></li></ol> 2。样品洗涤和干燥<ol><li>洗涤<ol><li>将每个头发样品放入15毫升螺旋盖聚丙烯离心管中。 </li><li>加入5 ml的高效液相色谱（HPLC）级异丙醇每个试管接着重复反演利用旋转3分钟。 </li><li>倒出异丙醇到废物容器中，以C不想失去任何样品。 </li><li>重复步骤2.1.2和2.1.3一次。 </li></ol></li><li>烘干<ol><li>干燥的头发，至少2-3天，以确保完整的异丙醇挥发。 </li></ol></li></ol> 3。试样磨CORT提取 - 方法1的大样本<ol><li>试样磨<ol><li>将高达250毫克的干发到10ml的不锈钢研磨以及一个12毫米的不锈钢研磨球罐子。 </li><li>研磨样品进行6分钟，在25赫兹的使用球磨机的速度。 </li><li>重达在分析天平上，将50mg的粉末状的头发，然后将其转移到一个干净2.0毫升聚丙烯微量离心管中进行后续CORT萃取。 </li></ol></li><li> CORT提取<ol><li>加入1.0毫升HPLC级甲醇含有粉末样品的离心管。 </li><li>盖上试管并在室温下与共同孵育样品18〜24小时nstant反转使用旋转器。 </li><li>离心管中，14,000 rpm离心1分钟，在室温下沉淀的粉末状毛发。 </li><li>转让0.6毫升上清液到一个干净的1.5 ml离心管，注意不要扰乱头发粉颗粒。 </li></ol></li></ol> 4。试样磨CORT萃取 - 方法2的小样本<ol><li>试样磨<ol><li>将高达60毫克的头发到一个预先称重2毫升离心管中增强胎圈跳动。 </li><li>再次称重小瓶以获得样品的重量。 </li><li>加33.2毫米铬钢珠到每个小瓶中，然后研磨样品的至少2分钟的珠打浆机。如果目视检查发现，该样品被充分磨碎，然后0.5或1分钟执行额外的研磨。注:在地面头发没有转移是必要的，这种情况下，因为CORT提取是在同一小瓶中进行研磨。 </li></ol&gt; </li><li> CORT提取<ol><li>加入1.5毫升的HPLC级甲醇中含有的粉末样品的离心管中。 </li><li>盖上管并使用旋转器在室温下以恒定反转孵育样品18〜24小时。 </li><li>离心管中，10,000 rpm离心5分钟，在室温下沉淀的粉末状毛发。注意:铬钢珠在萃取和离心步骤，留在管子与毛发，占下离心速度相比，方法1。 </li><li>将1.0毫升上清液到一个干净的1.5 ml离心管，注意不要扰乱头发粉颗粒。 </li></ol></li></ol> 5。溶剂蒸发和重构示例<ol><li>干下来使用一个真空蒸发器或氮气流中，如果在真空蒸发器是不可用的甲醇。如果在真空蒸发器中被使用时，甲醇蒸气可以借助于被捕获的冷阱或配备活性炭盒中的化学陷阱。 </li><li>下列除去甲醇，重构该CORT提取物中的EIA测定缓冲液（测定稀释剂）的适当体积。如果相对高CORT值预期的，然后用0.4毫升或更高的缓冲液体积。如果相对低的值是预期的，则可以将音量降低到0.2毫升或更少，以增加灵敏度。注意:0.2毫升是足够的体积，以在发生至少两次运行示例的重复的等分试样的重新执行是必要的。 </li><li>或者立即测定重构的样品或在-20℃下用于以后的分析冻结。注意:如果重组后的样品被冻结，以避免在由于样品体积的改变冻结期限升华重要的是会导致在最终的计算进行虚假CORT值。 </li></ol> 6。 CORT含量和数据转换<ol><li>测定头发中提取的CORT使用高灵敏度酶免疫测定（EIA）试剂盒。注意:如果使用的是商业唾液皮质醇EIA试剂盒，按照试剂盒插入不同的是测试样品将是每一个重组的头发提取物的重复等分，而不是唾液样本作为指定制造商的建议。 </li><li>准备一个质量控制（QC）毛发提取物在每测定法中使用。 <ol><li>收集了一些多余的毛发样本，并单独处理它们按照步​​骤4.1和4.2或泳池他们为大样本的处理步骤3.1和3.2。 </li><li>蒸发溶剂，并重构该提取物在步骤5.1和5.2，然后汇集从几个单独或混合样品重构提取物。 </li><li>分装汇集重构提取物（ 例如 0.10毫升）的一个适当的量成单独的微量离心管中，并冷冻供以后使用。 </li><li>解冻并一式两份每个免疫分析微孔板运行一个QC等分试样，以提供参考资料，希望能帮rence CORT值检查每个运行的质量。 </li><li>的变化（CV;定义为标准偏差由一组样本值的平均值的比率）的批内系数可以通过分析10-12 QC井在相同的运行来计算的，而一个批间CV可以计算用在一组运行的QC值。如果一个人的酶标仪软件算出的CV值表示每个毛发提取的复孔，然后用于确定批内CV的另一种方法是计算在整个板的那些个体的CV值的平均值。 </li><li>如果任何样品提取得到比在EIA最高CORT标准一读越大，则提取的另一等分试样稀释用的测定缓冲液适当量和随后的EIA读数被校正为稀释因子。样品还定期重新分析，如果CV为复孔是&gt; 10％。 </li><li>因为CORT环境影响评估套件是设计用来测量CORT VA梅毒液体样品，如唾液或血浆中，酶标仪软件的输出必须转换成皮质醇的量，每单位重量的粉状头发。下面的公式转换微克/分升，以每毫克头发皮克CORT检测输出: （A / B）*（C / D）* E * 10,000 = F 其中A =微克/分升的检测输出，B =体重头发进行提取（以mg），C =第一卷。甲醇（以毫升）加入到粉末状毛发，D =第一卷。 （以ml）从甲醇提取物回收，随后进行干燥下来，E =第一卷。 （以毫升）用于重建的干提取物分析缓冲液;和头发皮质醇浓度的皮克/毫克F =最终值。 </li></ol></li></ol>

<ol>
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该作者有没有利益冲突申报。

上述的头发CORT手续简单执行，是相对便宜的，利用了现成的化学品，试剂和耗材，需要的设备，有一个例外，很可能是出现在一个典型的分析实验室。唯一的例外是一个研磨装置如球磨机或mini-beadbeater。我们注意到，一些研究小组讳言头发样品切成小碎片大约1毫米的长度为12，但根据我们的观察，我们建议进行研磨，而不是如果在所有可能切碎。在文献中的方法的变化的另一个领域涉及?...

CORT血浆，唾液，或偶尔在尿液或粪便的测量已被用作生理应激自Selye的发现HPA轴中应力的作用1的索引。虽然众多的论文已发表有关HPA活性，急性紧张的情况下，该领域已经阻碍了缺乏慢性生理应激的一种简单而可靠的指标。这个问题的产生是因为HPA的活动是受昼夜节律变化，可以通过环境干扰羞愧血浆和唾液两者屈服"点"的估计。尿和粪便样品，得到跨越若干小时到一整天在某些情况下，皮质醇和/或代谢物排泄的测量。收集使用任何这些矩阵可以提供CORT水平的粗略综合指数随时间的多个样品的，然而，没有这些方法提供了HPA活性和本学年的响应真正的长期指数干以慢性压力。 头发中测量CORT已经开始填补这一重要的需要在应力文学。几个实验室最初的研究表明CORT在人类毛发的存在，但是并没有调查是否毛发皮质醇水平改变应力2的函数。由于我们的实验室已经拥有多年在恒河猴HPA轴的各种社会和行为因素3的调控，我们开始着手建立和验证提取的恒河猴毛4和分析方法。基于的前提是血源性皮质醇缓慢和连续地加入到生长的头发，这种新的方法的目的是要使用的头发源性皮质醇水平的HPA活性的综合指数比周的期间，以月。 有几个方法上的挑战是在开发本议定书遇到的问题。首先，以往的研究表明，少量Øf循环CORT被排泄在汗和皮脂，因此可以涂在头发轴2的外侧。为了消除这种潜在的变乱，我们开发了在出现以除去外部CORT而对CORT的影响最小日益增长毛干中存在轻微的洗涤程序。因此，猴毛经受这个过程（ 即两个3分钟洗涤用异丙醇）丢失的总发CORT含量约为7-8％，而第三洗涤除去从样品4低于1％的类固醇。似乎是在人的头发更外部CORT，因为同样的过程从样品（K. Rosenberg和J.迈尔，未发表）的平均27％的总CORT含量除去。像猴毛，但是，额外的洗涤含有少得多CORT（约7％），比前两个洗。因此，无论从猴子和人发结果支持这一论点，大多数（如果不是全部）外部CORT，同时保持一个主要的压裂被删除内部毛基质内化CORT的。第二，我们的试验性研究还表明，磨削头发萃取前从样品显著增加CORT恢复，据推测是由破开的毛干的复杂的蛋白质基质，以及增加的表面积可用于溶剂渗透。两种不同的研磨方法被开发，每一个优点和缺点。方法1，使用球磨机，有生产最好的粉末的优点。然而，球磨机是一种相对昂贵的设备和产品，如果使用标准的研磨罐和球，它是能够磨削只有两个样本在时间的。小样本也难以利用球磨机用标准的研磨罐进行处理。方法2，其使用beadbeater，是不太有效的研磨能力。其结果是，平均CORT回收率低约10％，使用该方法相比，在球磨机（未发表数据）。另一方面，一个beadbeater是相当便宜比球磨机，16-24样品可以一次取决于模型被​​研磨，且该方法非常适合用于小样本。因为上面提到的差的恢复，最好是使用相同的磨削方法为一个特定的研究中的所有样本。 一旦头发样品已经被处理，它们是用甲醇和CORT提取的提取物是由一个敏感和特异的商用EIA试剂盒最初被设计来测量唾液皮质醇进行分析。提取和测定方法进行了部分通过展示，从猴毛样品提取物的连续稀释产生的环境影响评估的读数紧密平行正宗CORT标准获得的读数验证。然后，我们发现，头发CORT（除血浆和唾液皮质醇）是猴子的新住房小区4,5的行政强制拆迁的主要生活应激敏感。该PRESEN吨纸提供了经常使用在我们的实验室来处理人类和猴子的头发样本，以及从这些样本提取和分析CORT的方法的详细说明。

<table border="0" cellpadding="0" cellspacing="0" width="798">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">HPLC-grade isopropanol</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:119px;">A451</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">HPLC-grade methanol</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:119px;">A452</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Salivary cortisol assay kits</td>
			<td style="width:116px;">Salimetrics</td>
			<td style="width:119px;">1-3002</td>
			<td>See manufacturer&#39;s kit insert for information on assay sensitivity and specificity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-ml polypropylene screw-cap centrifuge tubes</td>
			<td style="width:116px;">Max Scientific</td>
			<td>10-9151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">1.5-ml Safe-Lock microcentrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:119px;">05-402-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">2.0-ml Safe-Lock microcentrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:119px;">05-402-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.0-ml XXTuff reinforced microvials</td>
			<td style="width:116px;">BioSpec</td>
			<td>330TX</td>
			<td>Use with mini-beadbeater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.2-mm chrome-steel beads</td>
			<td style="width:116px;">BioSpec</td>
			<td>11079132c</td>
			<td>Use with mini-beadbeater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">10-ml stainless steel grinding jars</td>
			<td style="width:116px;">Retsch</td>
			<td style="width:119px;">02.462.0061</td>
			<td>Use with mixer mill</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-mm stainless steel grinding balls</td>
			<td style="width:116px;">Retsch</td>
			<td style="width:119px;">05.368.0037</td>
			<td>Use with mixer mill</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Savant activated carbon cartridge</td>
			<td style="width:116px;">Fisher</td>
			<td>DTK120R</td>
			<td>Use with Savant chemical trap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;"></td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;"></td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Name of Equipment</td>
			<td style="width:116px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rotator for 15-ml centrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td>S02135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rotator for microcentrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td>NC9854190&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benchtop centrifuge for microcentrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">MM 200 mixer mill</td>
			<td style="width:116px;">Retsch</td>
			<td style="width:119px;">20.746.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-Beadbeater 16&#160;</td>
			<td style="width:116px;">BioSpec</td>
			<td>607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Savant DNA Speedvac</td>
			<td style="width:116px;">Fisher</td>
			<td>DNA120-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Savant refrigerated vapor trap</td>
			<td style="width:116px;">Fisher</td>
			<td>RVT400-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Savant chemical trap</td>
			<td style="width:116px;">Fisher</td>
			<td>SCT120</td>
			<td>Alternative to refrigerated vapor trap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate washer</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate mixer</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Multichannel pipetter</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Analytical balance</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction and analysis of cortisol from human and monkey hair

应激激素皮质醇（CORT）正在慢慢融入人类，非人灵长类动物和其他哺乳动物的生长的头发轴。我们开发并验证了CORT的提取和分析，从猕猴毛发的方法，并随后与人类头发适合使用这种方法。相反，从血浆或唾液获得CORT"点样"，头发CORT提供了一个集成的测量下丘脑 - 垂体 - 肾上腺皮质（HPA）系统的活性，从而生理应激，激素过程中掺入的时期。因为人的头皮头发生长以1厘米/月的平均增长率，从头发段获得CORT水平长度数厘米有可能成为应激经历了数月的生物标志物。 在我们的方法中，每个头发样品首先洗涤两次，在异丙醇中，以从已经沉积的汗或皮脂发轴的外侧除去任何CORT。干燥后，将样品研磨成细粉末，打破了头发的蛋白质基质和增加表面积进行萃取。 CORT从毛发轴的内部被提取到甲醇中，将甲醇蒸发，将提取物重新溶解在测定缓冲液中。提取CORT，随着标准和质量控制，然后由一个敏感和特异的市售的酶免疫测定（EIA）试剂盒进行分析。从环评读数转化为每毫克粉末状毛重量皮克CORT。这种方法已经被用在我们的实验室进行分析头发皮质醇的人类，几种猕猴，狨猴，狗和北极熊。无论是从我们的实验室和其他研究小组已经有很多研究表明头发CORT的广泛适用性评估慢性应激暴露在自然以及实验室的设置。

图1显示了一组人的头发样本（成年男性和女性人类受试者）的代表打印输出使用方法2研磨和萃取处理。计算机软件被用来产生数据输出，并以适合的4参数S形曲线的皮质醇水平（ 图2）。从这个板孔之间的简历介乎0.01-5.73％与1.34％的平均批内CV。来自九个最近的人的头发化验用的QC值是4.41％，批间CV测定。在这个板块分析的37个​​样品产生了一系列从3.1-650皮克/毫克的?...

Watch this Scientific Journal Video about Extraction and Analysis of Cortisol from Human and Monkey Hair at JoVE.com

Extraction and Analysis of Cortisol from Human and Monkey Hair

皮质醇（CORT）积聚在人类和非人类灵长类动物的生长的头发轴。我们描述了用于提取和分析头发CORT以高精度和灵敏度的方法。是特别适合用于评估慢性压力过周时期到几个月头发CORT的测量。

提取与分析皮质醇从人与猴的毛

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

extraction-and-analysis-of-cortisol-from-human-and-monkey-hair

Research

JoVE Journal

Biology

28.1K 次观看.  University of Massachusetts, Amherst. 皮质醇（CORT）积聚在人类和非人类灵长类动物的生长的头发轴。我们描述了用于提取和分析头发CORT以高精度和灵敏度的方法。是特别适合用于评估慢性压力过周时期到几个月头发CORT的测量。 

视频: Extraction and Analysis of Cortisol from Human and Monkey Hair

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 ...

科研

生物学

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

基因功能分析和可视化的纤毛产生的流体流动的在枯否囊泡中

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

提取毒液和毒腺Microdissections从蜘蛛的蛋白质组学和转录组分析

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

新技术脱发的定量分析小鼠使用灰度分析

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法:从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...