Name: tissue collection of bats for -omics analyses and primary cell culture
Uploaded: 2019-10-23T13:00:00
Description: Watch this Scientific Journal Video about Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture at JoVE.com

我们感谢CEBIO、埃里卡·帕利扎、米卢斯卡·桑切斯、豪尔赫·卡雷拉、埃德加·伦吉福·巴斯克斯、哈罗德·波罗卡雷罗·扎里亚、豪尔赫·鲁埃兹·莱沃、海梅·佩切科·卡斯蒂略、卡洛斯·特洛、范尼·科内霍和范尼·费尔南德斯·梅洛制作纸巾收集在秘鲁可能。我们还感谢JaraguaGrupo Jaragua和Yolanda León的所有成员在多米尼加共和国进行组织采集，并感谢伯纳尔·罗德里格斯·埃尔南德斯、伯纳尔·马塔里塔和哥斯达黎加拉塞尔瓦生物研究站的每个人采样可能。我们感谢Ella Lattenkamp和Lutz Wiegrebe为细胞培养生成提供从Phyllostomus变色蝙蝠的翅膀冲头的获取和样本收集。细菌变色蝙蝠起源于慕尼黑路德维希-马克西米利安大学生物系II的繁殖地。慕尼黑地区兽医办公室批准了饲养和繁殖蝙蝠。LMD、SJR、KTJD 和 LRY 由 NSF-DEB 1442142 提供资金。LMD 由 NSF-DEB 1838273 提供资金。LRY 由 NSF-PRFB 1812035 提供资金。SCV 和 PD 由马克斯·普朗克研究小组奖和人类前沿科学计划 （HFSP） 研究资助 （RGP0058/2016） 资助。SJR、JHTP和KTJD由欧洲研究理事会（ERC启动赠款310482[EVOGENO]）资助给SJR，ECT由欧洲研究理事会赠款（ERC-2012-StG311000）资助。

这里描述的所有方法都已获得石溪大学机构动物护理和使用委员会（IACUC）的批准（#2013-2034年协议）。
1. 安乐死
<ol><li>用异透麻醉剂或其他可用的麻醉剂润湿棉球。</li>
	<li>将棉球放入可重新密封的密封塑料袋中。袋子应该足够大，可以舒适地放在布蝙蝠袋中。</li>
	<li>几分钟后，让袋子中弥漫着麻醉剂，将装有蝙蝠的蝙蝠袋放入塑料袋中。</li>
	<li>等待几分钟，直到蝙蝠被...

本手稿中讨论的协议描述了蝙蝠各种高通量分子分析的最佳采样实践。所有成功的-组质研究都需要高质量的组织，但取样蝙蝠组织以及其他非模型生物，往往发生在无法设定为与受控实验室环境相同的标准的现场条件下。取样通常发生在偏远地区，资源很少，包括使用电力和冰柜的机会有限。很难，而且往往不可能确保完全无菌的取样条件。因此，概述的是在各种取样地点和现场条件下被确定为...

高通量测序 （HTS） 方法在效率上迅速提高，成本降低，现在可以将这些方法扩展到数百或数千个样本。这些技术的应用所获得的见解在从生物医学到进化和生态学1、2、3等多个科学学科中有着巨大的影响。然而，许多HTS应用严重依赖来自活源的高质量核酸。随着基于长读分子4的第三代测序的发展，这种限制可能会变得越来越成问题。出于这些原因，需要集中力量建立从实验室外的野生生物中收集新鲜组织样本的最佳做法，从而最大限度地发挥材料的效用，减少需要收集。
Bat1K是一个国际科学家联盟，目前正在将每种蝙蝠的基因组测序到染色体级集合5。蝙蝠代表了哺乳动物多样性的20%，具有特殊的适应，对理解衰老、疾病生态学、感官生物学和新陈代谢有影响。许多蝙蝠也由于人类剥削而受到威胁或濒危，或由于病原体8、9而迅速减少，基因组测序对于保护这些物种非常重要。尽管 Bat1K 目前的目标是对所有蝙蝠物种的基因组进行测序，但为高质量基因组测序收集组织样本的标准化仍然是整个生物体生物学家群体面临的一项关键挑战。除了基因组数据外，对蝙蝠适应多样性的功能理解还需要组织特定的转录组和蛋白质分析，通常需要单独的采集方案。此外，与所有分类组一样，虽然最佳组织收集和保存对于获得最高质量的数据进行-组学分析至关重要，但由于技术和快速变化，交流最佳做法往往很困难。多个研究团队独立工作。
鉴于许多蝙蝠物种很少或受到威胁，需要采用蝙蝠-组学研究的最佳做法尤为迫切。与其他小型哺乳动物（如啮齿动物和精明动物）不同，蝙蝠寿命长，由于特殊的DNA修复机制10和缓慢繁殖11，大多数物种每年只产一只或（少数）两只幼。由于这些原因，蝙蝠种群可能缓慢地从干扰中恢复，从野外采集许多个体既不可取，也不可行。换句话说，必须优化协议，以获得单个样本的最大数据量，从而减少不必要地复制采样工作的需求。
在这里，该协议特别侧重于用于基因组和转录组测序和蛋白质分析的蝙蝠组织的收集和采样的标准化方法。其最高优先事项是确保蝙蝠组织以合乎道德和负责任的方式收集，从采集许可程序、组织出口到尽量减少对动物的压力，以及长期储存条件。精心剖面，目的是面向未来，证明所收集不同材料的有用性。本手稿提供了一个分步指南，以人道的方式收集蝙蝠，旨在尽量减少对人口的影响，并最大化科学价值。虽然该协议的重点是专门用于蝙蝠，但许多步骤是相关的其他脊椎动物分类，特别是哺乳动物。
组织收集概述 组织收集程序，包括储存温度和选择保存剂，将由计划的任何下游分析的性质决定。然而，强烈建议在可能的情况下，在一系列方法下收集组织，以最大限度地发挥其未来效用，即使没有计划进行具体分析。一般来说，组织被收集和保存，以便随后分析核酸（DNA和RNA）或蛋白质。对于每种应用，通过直接在液氮 （LN2） 中快速冻结，可以最佳地保存组织。然而，在现场并不总是能够立即浸入 LN2。随着技术的进步，在环境温度下储存DNA和RNA的专用小瓶等资源越来越容易获得。虽然我们还没有验证本协议中的所有此类材料，但我们鼓励其他研究人员相对地分析新材料相对于我们这里的性能。在无法访问 LN2 的情况下，例如，由于在亚马逊的小型平面进行站点访问而无法进行 LN2 传输时，我们确实提供了为不同应用提供理想的保存组织的方法。此外，我们还提供了一种收集组织的方法，从中可以生长和繁殖活细胞。下面我们概述了为上述每个目的收集材料的关键注意事项，表1概述了收集方法。
脱氧核糖核酸组织 对于采集的所有采集组织，存储介质将确定其是否可用于标准或高分子量 （HMW） DNA 提取。HMW 是长读测序所必需的，目前需要生成染色体级基因组组件或"白金标准"基因组。低分子量 （LMW） DNA 可以从闪存冷冻、AllProtect（因此称为"组织稳定溶液"），甚至 RNAlater（因此"RNA 稳定溶液"）保存的样品中提取（尽管闪光冷冻样品保持最佳).通过标准实验室方法（例如，硅胶膜旋转柱、苯酚氯仿）分离的DNA仍可能产生高达20千基（kb）的DNA片段。因此，只要有足够的产量，这种形式的分离DNA可用于单刀片大小库制备，其中插入大小通常为+500基对（bp），并生成±100 bp的短序列读取12。这种DNA对于不需要全长染色体数据的"重新测序"项目或研究特别有用。HMW DNA （10-150 kb） 更具挑战性，只能使用收获后在 LN2 中快速闪光冻结的组织可靠地获得，并在-80°C中保持至-80°C，直到提取。
低分子量或支离破碎的DNA通常足以用于靶向方法，包括通过PCR和短读测序13进行基因扩增。基于PCR的研究使用LMWDNA，只针对一个或几个基因已经高度信息，了解适应和分子进化蝙蝠感官生物学6，14，生理学15，植物遗传学5，16，和保存17，18。成功靶向序列重获低分子量和碎片DNA也已证明为许多脊椎动物群，包括蝙蝠19。这些方法通常具有成本效益，对蝙蝠具有微微侵入性，因为粪便样本和非致命组织通过拭子或翼活检打孔进行取样也是获得低分子量分析DNA的常用方法。 21.
但是，质量在很大程度上取决于存储样本的介质类型22。经过对buccal拭子和活检冲孔的系统定量比较，机翼活检液的拳头已经证明能持续产生更高的DNA水平，在采集22过程中对蝙蝠的压力较小。这些比较还表明，当翼冲剂保存在指示器二氧化硅（即一种由硅胶珠制成的干燥剂，在观察到水分时改变颜色）时，而不是在其他流行的储存介质（如乙醇或DMSO22;但是，其他存储介质，包括组织稳定溶液没有被检查。翼冲也可用于在培养中生长成纤维细胞，如在Kacprzyk等人23和下述（见第6节）。对于这些方法，应轻轻扩展机翼或尿毒症，并应使用直径为 3 mm 的清洁活检冲床来获取样品。这种方法似乎不会造成持久的伤害，在大多数情况下，疤痕在几周内愈合。
HMW DNA （10-150 kb） 更具挑战性，目前仅使用收获后在 LN2 中快速闪光冻结的组织获得可靠，并在 -80°C 中保持至 -80°C，直到提取。HMW DNA （10-150 kb） 对于长期读取的 DNA 测序至关重要，因此对于 de novo 基因组组装至关重要。事实上，虽然大多数商业试剂盒可用于分离一些标准的HMW DNA，但产生的分子尺寸往往不符合第三代测序技术的要求，例如太平洋生物科学（PacBio）等公司推出的技术，牛津纳米孔技术，和10x基因组学，或通过生物纳米基因组学或多尾基因组学提供的组装方法*。因此，对"超 HMW"DNA（>150 kb）有新的需求。当从蝙蝠获得超HMWDNA时，肝脏、大脑或肌肉的新鲜样本都适合，但这些样本必须立即在LN2中闪光冷冻，没有任何存储缓冲液或冷冻保护剂。这些步骤的完整描述超出了本文的范围，但可在其他部分25提供。
RNA组织 RNA是一种单链分子，其稳定性低于DNA。虽然有多种形式的RNA，-组分分析往往侧重于mRNA（信使RNA）和小型RNA（例如微RNA）。转录后，mRNA被拼接成一个成熟的转录本，其中不包含内创体，并代表基因/基因组的编码部分。编码基因只占基因组大小的一小部分（1%-2%），使靶向mRNA成为获取基因序列数据的具有成本效益的方法。微RNA是一类RNA，用于调节mRNA转化为蛋白质的过程，因此是重要的调节效应者。RNA转录本可以单独排序，或者更常见地用于-omics分析26，27，28，29，30，作为转录本的一部分;即给定样本中存在的所有RNA转录本的总数。
排序可以按照多种方法执行（即，通过短读RNA-seq或长读全异形Seq），允许分析RNA丰度和异形使用。由于mRNA转录本的数量和多样性在细胞和组织之间不同，RNA测序使得研究和比较不同样本的基因表达和调控成为可能。随着这些方法在生物学上越来越丰富，对小型RNA和全等形测序测序的兴趣正在增长。组织样本的制备，以序列不同类别的RNA可以执行的方式，如本手稿中介绍，只有随后的提取方法不同31，32。最后，由于转录组提供了蛋白质编码基因组的高覆盖率子集，因此组装的数据集在基因组组装和注释方面可能有用，因此跨一系列不同组织收集RNA-seq数据是Bat1K 计划。
与DNA相比，RNA在化学上不稳定，并且也是RNase酶的目标，这些酶在组织裂解物中普遍存在，作为对抗RNA病毒的防御策略。由于这些原因，细胞和组织中的RNA分数在取样和/或安乐死后不久开始降解。因此，保存RNA需要采取措施防止其降解。这通常涉及在稳定剂（如RNA稳定溶液）中保存4°C下新收集的组织，以灭活组织中自然存在的RNases，然后冷冻以进行长期储存。作为首选的替代方案，组织可在LN2中闪光冷冻;虽然如上所述，将LN2运送到现场并保持水平以防止组织解冻在后勤上可能具有挑战性。
蛋白质组织 蛋白质组成和相对丰度在细胞和组织之间以与RNA讨论的方式相似;然而，蛋白质平均比RNA更稳定。使用蛋白质组学进行蛋白质鉴定通常与蛋白质序列的一小部分（而不是整个蛋白质序列）相匹配，但它可以提供有关组织间表达的信息，并表征存在病原体。由于许多蛋白质序列在哺乳动物中保存，蛋白质组学的蝙蝠样本很容易被保存的人类蛋白质污染，在采集过程中需要无菌方案（例如手套、钳子）。虽然LN2中的闪冻结是防止蛋白质降解的最好方法，但如果没有其他方法，使用干冰、-20°C冰柜，甚至冰是合适的。随着温度的升高，蛋白质分裂的风险也会增加。组织稳定溶液等稳定剂在室温下有效保存组织的蛋白质部分，适合在闪光冷冻不可行时进行短期保存（最多一周）。
给定组织的酶轮廓直接影响其中蛋白质的保存。具有低酶活性的组织（如肌肉）即使在家庭冰柜的较高温度下也能保留蛋白质的轮廓。相比之下，肝脏组织是酶反应的，其蛋白质在制备过程中有更高的降解概率。越来越多的协议从甲苯固定石蜡嵌入（FFPE）样品获取人类蛋白质组谱，这表明，当采集时冷冻时，组织甲醛固定有望实现低成本蛋白质保存可行33，34.虽然高度依赖于保存时间和条件，蛋白质已经通过免疫性化学从形式固定，乙醇保存蝙蝠标本35鉴定。这种方法无法扩展到蛋白质组级采样，但突出表明，当闪存冻结不可用且其他稳定剂成本过高时，正式固定蝙蝠组织可能产生蛋白质配置文件。
细胞培养组织 取样组织和闪光冷冻提供了有限的材料可供使用，一旦材料被使用，它不再可用。或者，细胞培养物提供活细胞，可以立即用于或保存用于未来的研究。培养也促进细胞的膨胀，当组织样本较小时提高产量。在组织收集有限的情况下，它特别有用，例如对稀有物种进行实验，其中非致命性取样至关重要，因此对养护具有广泛影响。描述是一种方案，其中细胞培养可以通过翼膜组织的非致命采样，但培养是可能的与多种组织类型36，37。此处提供的协议为粘附单元选择。源组织和生长介质的组合使该协议适合选择和生长成纤维细胞，但如果需要，替代协议可用于选择其他细胞类型。在Bat1K项目的背景下，据预测，对于稀有和受威胁的物种，对翼膜进行非致命性取样和通过培养扩大样品对于产生采用多种技术所需的DNA数量至关重要。.
蝙蝠捕获 所有处理蝙蝠的人都应接受蝙蝠能力研究人员的培训，并接种一系列接触前注射疫苗，以预防狂犬病。如果被咬伤，还需要再注射一系列暴露后注射。捕捉蝙蝠的标准方法包括雾网（图1）和竖琴陷阱（图2）。雾网是最常用的，非常适合低到中度活动的地区，因为它们需要最小心，以尽量减少蝙蝠的苦恼。小蝙蝠特别脆弱，如果不迅速死亡，可能会因压力而死亡。频繁的净检查最大限度地减少了蝙蝠伤害和死亡率以及雾网损坏。这个细节很重要，因为适当的组织收集需要组织是新鲜的，对蝙蝠的雾网的不当关注可能导致不必要的死亡或过早死亡，研究人员才能正确处理样本。由于几只蝙蝠可以以最小的痛苦在竖琴陷阱中休息，因此这种方法非常适合蝙蝠活动量高的区域，如洞穴附近或大型栖息区。补充方法中提供了关于适当捕获蝙蝠和数据处理以收集形态和人口信息的详细说明。

<table>
	<tbody>
		<tr>
			<td>1.5 mL Eppendorf Safelock tubes</td>
			<td>Fischer Scientific</td>
			<td>10509691</td>
			<td>1.5 mL sterile tubes used during cell culture protocol</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Sarstedt</td>
			<td>62,554,002</td>
			<td>15 mL sterile tubes used during cell culture protocol</td>
		</tr>
		<tr>
			<td>2 mL cryovials</td>
			<td>Thomas Scientific</td>
			<td>1154P75</td>
			<td>cryogenic vials for tissues to be stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>3-mm biopsy punch</td>
			<td>Medline</td>
			<td>MIL3332</td>
			<td>wing biopsy punch for cell culture</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Greiner</td>
			<td>83,392</td>
			<td>Culture vessle</td>
		</tr>
		<tr>
			<td>Allprotect Tissue Reagent</td>
			<td>Qiagen</td>
			<td>76405</td>
			<td>for fecal samples; tissue stabilizing solution</td>
		</tr>
		<tr>
			<td>Cell counting slides for TC10/TC20 cell counter, dual chamber</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
			<td>Chambers for the count cells using the automated cell counter TC20 by Bio-Rad</td>
		</tr>
		<tr>
			<td>collagenase IV</td>
			<td>Stemcell Technologies</td>
			<td>7909</td>
			<td>For dissociation of primary cells</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650-5X5ML</td>
			<td>Prevents crystalization of water during freezing of the cells.</td>
		</tr>
		<tr>
			<td>DMEM&#8211;Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher</td>
			<td>12491-015</td>
			<td>culture media</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td>Balanced salt solution used for washing cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Fischer Scientific</td>
			<td>10270106</td>
			<td>Serum-supplement for in vitro cell culture of eukaryotic cells</td>
		</tr>
		<tr>
			<td>Gentamycin sulfate salt</td>
			<td>Sigma Aldrich</td>
			<td>G1264-250MG</td>
			<td>Antibiotic for culture media</td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty</td>
			<td>Thermo Scientific</td>
			<td>5100-0050</td>
			<td>Freezing container which provides 1&#176;C/min cooling rate</td>
		</tr>
		<tr>
			<td>PARAFILM</td>
			<td>Sigma</td>
			<td>P7793</td>
			<td>Wrapping tubes etc for sealing</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (pen-strep), 100x</td>
			<td>Invitrogen</td>
			<td>15140130</td>
			<td>Antibiotic for culture media</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) 10x, pH 7.4</td>
			<td>Thermo Fisher</td>
			<td>10010023</td>
			<td>salt buffer used for washes and storage of bone tissue; dilute to 10X using de-ionized water</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermo Fisher</td>
			<td>AM7021</td>
			<td>RNA stabilizing solution</td>
		</tr>
		<tr>
			<td>RNAse away</td>
			<td>Genetech</td>
			<td>83931-250mL</td>
			<td>breaks down enzymes that lead to RNA degradation</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Fisher Scientific</td>
			<td>7631-86-9</td>
			<td>dessicant agent</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td>1450102</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Bio-Rad</td>
			<td>145-0013</td>
			<td>Cell stain used to assess cell viability</td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4049</td>
			<td>For dissociation of cells during splitting</td>
		</tr>
	</tbody>
</table>

tissue collection of bats for -omics analyses and primary cell culture

随着高通量测序技术的进步，高质量组织采集和保存的标准化方法允许将这些方法扩展到非模型生物体。为一系列高通量测序方法开发了一系列优化蝙蝠组织收集的方案。这里概述了捕获蝙蝠的协议、为每个蝙蝠收集所需的人口统计数据，以及优化方法，以在组织采集过程中将蝙蝠的压力降至最低。具体概述的方法用于收集和治疗组织，以获得 （i） 用于高分子量基因组分析的 DNA，（ii） 组织特异性转录组RNA和 （iii） 蛋白质组级分析的蛋白质。最后，还概述了一种通过从翼夹创建可行的原细胞培养物来避免致命采样的方法。这些方法的一个中心动机是最大化每只蝙蝠的潜在分子和形态数据量，并提出保存组织的最佳方式，以便它们随着新方法在未来的发展而保持其价值。随着世界各地出现了对染色体级、无误差的物种基因组进行测序的倡议，这种标准化变得尤为重要，其中多个科学方正在带头对不同的分类学进行测序。组。此处概述的协议定义了 Bat1K 的理想组织收集和组织保存方法，该联合体负责对每种蝙蝠的基因组进行测序。

Dna 对于标准低分子量 （LMW） 分析，从三个新热带蝙蝠物种中提取了 DNA。根据本文件所述的协议，从多米尼加共和国和哥斯达黎加的实地采集了组织样本。解剖后，将混合组织（脑、肝、肠）的小块（最薄部分为<0.5cm）放入RNA稳定溶液中，闪速冷冻，然后储存在-80°C。提取使用标准DNA提取协议与RNase处理43。使用自动电泳工具评估DNA完整性...

Watch this Scientific Journal Video about Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture at JoVE.com

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

这是一种方案，用于对在野外捕获的蝙蝠进行基因组、转录组和蛋白组分析的最佳组织制备。它包括蝙蝠捕获和解剖、组织保存和蝙蝠组织细胞培养的协议。

蝙蝠组织收集 -细胞分析和原细胞培养

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

在这段视频中，我们提供了以人道方式收集蝙蝠的分步指南，旨在最大限度地减少对种群的影响，并最大限度地提高科学价值。我们的方法的优点是，我们最大限度地利用每只蝙蝠的潜在分子和形态数据量，保存组织以保留其未来价值，并最大限度地减少野生个体的收获。解剖协议很难口头证明。我们在这里介绍的许多组织准备技术最好通过可视化不同的器官是如何被识别的，然后正确解剖和储存来传达的。在开始任何解剖之前，请准备好所有小瓶，以避免延迟。对于 RNA，留出每个标本所需的小瓶数量。用要收集的组织类型以及标准标本识别信息标记每个管。将小瓶装满RNA稳定溶液50%，冷却至4摄氏度。注意不要用RNA稳定溶液完全填充小瓶，因为管子在液氮中放置时可能会爆炸。安乐死后，立即对蝙蝠标本进行斩首，如文本协议所述。从头发、筋膜和头骨肌肉（包括鼻子上的皮肤）中剥下头骨皮。小心不要打断鼻子的前端。用钳子去除眼睛，用足够强大的力分离视神经。将眼睛放在两毫升的RNA稳定溶液中。用剪刀从颈部开始，在头骨的后部做一个下垂的切口，注意不要损害大脑。然后，用钳子轻轻地拉回头骨的两侧，直到它裂开，露出大脑。确保颅骨的头骨已被切除，以便钳子可以轻易地刮走大脑。用钳子轻轻刮脑。大脑会很软。嗅球将变得可见，坐在头骨内部的腹部。尽量保持嗅觉灯泡连接。如果可能，保持大脑形状完好无损，并立即放在干冰上，或放在五毫升的RNA稳定溶液中。在头骨的头骨端，现在应该在头部的每一侧横向可见。使用钳子轻轻拉取科利。然后，将它们放入一个两毫升的RNA稳定溶液中。做两个切口，顶部和底部下颚连接，并删除下颚。克里布里形板将变得明显。这是保持研究人员导向的关键骨头。它可以被确定为头骨的最前部区域，有多个前身，有嗅球的两个凹槽。一旦下座被移除，从头骨的剩余部分取下讲台。确保钳口包括 cribriform 板。将鼻子放在RNA稳定溶液中，并储存在4摄氏度过夜。从下层，用剪刀切舌头，并放在两毫升小瓶的RNA稳定溶液。将鼻瓶放在液氮中，在RNA稳定溶液中过夜后。在冰上或冰箱中浸泡24小时后，将样品放入液氮中。使用手术刀刺穿腹腔，使纵向切口到肋骨。这会丧失骨骼框架。剥去皮肤以露出胸肌，并至少采集两个肌肉样本。在这些解剖过程中，会收集RNA和DNA的组织。将高分子量DNA的组织放入干低温中，并在液氮中闪冻。将RNA组织放在RNA稳定溶液中。切开胸骨，把肋骨拉开。现在，取回肺和心脏。使用手术刀和钳子将心脏与肺分离。使用手术刀分解肺部，并放在RNA稳定溶液中。收集心脏，这可以采取整体，但应切成两半，使RNA稳定溶液彻底浸泡。现在，从肝脏中抽取样本。至少采集两个肝脏样本。将一个样品放入小瓶中，以冷冻高分子量DNA，将另一个样品放入RNA稳定溶液中。沿着肝导管找到胰腺和胆囊。将胆囊转移到一小瓶RNA稳定溶液中。找到胃，和羽毛状的脾脏在它的基础，显示为不同的紫色阴影。胰腺也应该在这里作为重量结构可见。收集胃，脾脏和胰腺，在RNA稳定溶液各自的小瓶。收集小肠和大肠的小样本。放在RNA稳定溶液的各瓶中。拿一个肾脏，并跟随他们的导管到膀胱，然后放在各自的小瓶RNA稳定溶液。使用其他肾脏作为指南，找到睾丸，如果男性，或子宫和卵巢，如果女性。如果可能，收集一个或两个性腺。也采取翅膀皮肤的不同部分的样本，并保持在单独的小瓶。我们方法的主要优点是，它允许我们以非致命性的方式对动物进行采样，同时仍然为基因组研究以及功能分子研究生成足够的材料。我们的协议允许从翅膀组织制备原发性成纤维细胞。这些细胞是遗传物质的珍贵可再生来源，也是蝙蝠功能和分子探索的模型。根据书面协议第六节，在生长介质中保存的翼形夹子应转移到组织培养设施。将管内物转移到15毫升锥形离心管中。翼膜活检用于此演示。小心地去除生长介质。用500微升无菌PBS轻轻清洗活检两次。向管子中加入500微升，每毫升一毫克胶原酶四。这会导致组织消化成单个细胞。在37摄氏度下孵育过夜，无搅拌。第二天，制作新的生长介质，并预热至37摄氏度。准备一个六井组织培养板，在要使用的每个井中加入两毫升的新鲜预预警生长介质。将此板存放在 37 摄氏度和 5% 的二氧化碳培养箱中，直到需要。从培养箱中取出含有细胞的15毫升管，加入一毫升新鲜生长介质，抑制消化反应。通过小心地用P1000移液器尖端对溶液进行三叶化，实现单个细胞悬浮，重新悬浮细胞。在桌面离心机中轻轻旋转电池三分钟，以300倍G.用P1000移液器轻轻去除80至90%的液体，丢弃上清液。将颗粒重新在500微升的预预警生长介质中。轻轻三角悬浮，以确保颗粒不再可见，细胞被充分悬浮，确保避免小块组织，这些组织可能仍然可见在墙上。轻轻地将整个细胞悬浮液体积移液到先前准备的六井板的单一井中，其中包含预热生长介质。从一侧到一侧，从正面到背面轻轻摇动板两到三次，帮助细胞在单个层中的井面上分布。在显微镜下检查镀块。它们应该是出现球和浮动的单个细胞，但非常密集。小心地将板放入孵化器中，预设为 37 摄氏度和 5% 的二氧化碳。大约24小时后，在显微镜下观察细胞，以确定培养者的健康。单元格现在应附加到板表面，并显示为扁平化。将加湿培养箱中的细胞预设为37摄氏度和5%二氧化碳。应定期在显微镜下观察细胞，以确定需要介质茶点或分裂。必要时，小心地从井中吸入约50%的介质，并轻轻地在井边加入一毫升预预热生长介质，以免干扰细胞。当需要通过细胞时，小心吸气约90%的生长介质。将一毫升无菌PBS加入井壁，非常温和地清洗细胞，以免干扰细胞。来回轻轻摇动板，并侧摇两到三次。在再次清洗细胞之前，请小心地从盘子中吸出所有 PBS。将 trypsin EDTA 添加到井中，轻轻摇动板，用 trypsin EDTA 覆盖井的整个表面，并在室温下孵育一分半钟。通过添加一毫升新的预警告生长介质来抑制反应。上下移液约五次，从板表面清洗细胞，并确保细胞悬浮。将细胞悬浮液放在15毫升的管子中，在桌面离心机中旋转3分钟，达到G.丢弃上清液，用P1000移液器轻轻去除80%至90%的液体。将颗粒重新悬浮在一毫升预警告生长介质中，并轻轻三角悬浮液。确保颗粒的颗粒或大片段不再可见，并且细胞在单个细胞悬浮液中。要使用自动细胞计数器对细胞进行计数，请将悬浮细胞的十微升等同体与 trypan 蓝色混合在一起，以检测可行的细胞。孵育一分钟，将溶液的移液器10微升放到计数幻灯片上。将计数幻灯片插入自动单元格计数器，使用适当的设置对单元格进行计数。通过将生长介质与 DMSO 相结合，准备冷冻介质，以获得 10% DMSO 的最终浓度。颗粒应从六井板的 80 到 90% 汇合井中准备。将颗粒重新在一毫升半的冷冻介质中。将大约750微升的细胞悬浮液放在两个独立的冷冻中。将小瓶放在低温冷冻容器中，使细胞缓慢冻结，以保持细胞活力。立即将它们放在零下80摄氏度。要解冻冷冻细胞，准备一个六井板，其中包含两毫升预谋生长介质，每一个井将被使用。将板在37摄氏度和5%的二氧化碳培养箱中保持，直到需要。从液氮中吸收一小瓶细胞，放在温水浴中，快速解冻细胞。这需要两到三分钟。一旦小瓶中的溶液解冻，轻轻上下移液器使溶液均质化，并立即将整个溶液放在六井板的一个井中。从一侧到一侧，从正面到背面轻轻摇动板两到三次，帮助细胞在单个层中的井面上分布。将细胞在37摄氏度和5%的二氧化碳下放置24至48小时。监控和通过细胞，如证明。对于三种蝙蝠的标准DNA分析，DNA提取的代表性结果显示出来。单个大带表示最小碎片，以及每个提取的DNA碎片大小相似。RNA提取成功的一个常见指标是RNA完整性数（RIN），其值高于8表示触觉RNA中的高质量。这里展示的是从13种不同种类的新热带蝙蝠物种中提取RNA的RIN值，在四个不同地点取样。从安迪、哥斯达黎加和多米尼加共和国的物种中取样的组织在一夜之间浸泡在4摄氏度的RNA稳定溶液中后，直接置于液氮中。从亚马逊物种中取样的组织在放置RNA稳定溶液大约一周后被放入液氮中，并连续保持在4摄氏度。即使在这种情况下，RIN 值也可与直接放入RNA稳定溶液中的值相当，并直接放入液氮中。在组织培养之后，蝙蝠细胞培养物应覆盖80%至90%的板面，并保持其原始形态。这表示拆分的最佳阶段。经过六次超过六次的通道后，细胞将改变其形态，变得更大和更长，细胞将进入衰老。明亮的球向上的细胞代表死细胞。培养中的细胞代表可再生的物质来源，如DNA、RNA和蛋白质。这些细胞系可以冻结，从而提供一种资源，可用于多年的研究。这允许基因组学、转录学和功能学研究，使用原组织将具有挑战性。例如，细胞可以通过基因或化学方式进行改造，以观察对细胞表型的影响。

tissue-collection-of-bats-for-omics-analyses-and-primary-cell-culture

Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

静电纤维聚合物支架用于组织工程和细胞培养

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

酵母核内体系统的超微结构分析的纳米金标记

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

雌激素原代牛颗粒细胞的组织培养模型

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...