这项工作得到了NEI R21EY033941（对吴洪利）的支持;国防部W81XWH2010896（致吴洪利）;R15GM123463-02 （致Kayla Green和Hongli Wu）

所有动物实验均按照视觉和眼科研究协会关于在眼科和视觉研究中使用动物的指南进行。程序批准由北德克萨斯大学健康科学中心动物护理和使用委员会（协议编号：IACUC-2022-0008）授予。这些研究使用了通常低于2周龄的年轻C57BL / 6J小鼠。
1.培养基制备及晶状体解剖
<ol><li>通过向 450 mL DMEM 中加入 50 mL 胎牛血清 （FBS） 和 0.1 mL 50 mg/mL 庆大霉素来制备培养基。</li>
	<li>人道地对小于2周的C57BL / 6J小鼠实施安乐死。 注意：我们使用 CO2 吸入法实施安乐死。CO2 安乐死系统的最佳流速应位移 30% 至 70% 的腔室或笼容积/分钟。</li>
	<li>用手术剪刀轻轻取下眼睑，并用弯曲的镊子在眼窝的两侧轻轻按压，使眼睛向外突出。使用白内障刀在角膜上仔细切开，然后用弯曲的镊子小心地取出晶状体，确保不会损坏晶状体或其囊。 注意： 执行这些步骤时要小心，以保持镜头囊的完整性。由于镜片的脆弱性，使用带有弯曲和钝头的解剖工具非常重要，以最大限度地降低镜片损坏的风险。</li>
	<li>使用带有钝头的弯曲镊子将镜片转移到 60 mm 塑料组织培养皿中，该培养皿中装满了 5 mL 预热和无菌的 Dulbecco 磷酸盐缓冲盐水 （DPBS） 溶液，其中含有 10 μg/mL 庆大霉素。</li>
	<li>用含有 10 μg/mL 庆大霉素的 DPBS 溶液轻轻冲洗镜片，以去除任何潜在的碎屑或污染物，为进一步处理镜片做好准备，并保持无菌培养环境。</li>
	<li>为了获得足够数量的 LEC，将 4 个透镜用于 24 孔培养板，将 6 个透镜用于 6 孔培养板。</li>
</ol>2. LECs隔离
<ol><li>完成漂洗过程后，将镜头放在一张滤纸上，让它干燥。</li>
	<li>镜片充分干燥后，小心地将其转移到培养皿的盖子上，为取出镜片胶囊做准备。</li>
	<li>向上旋转晶状体，确保前节朝上。在使用镊子夹住前囊的同时，使用惯用手的囊镊子在囊中产生一个小撕裂。向相反方向轻轻拉动两个工具以取出胶囊并将其放入 DPBS 中，直到完成所有晶状体解剖。 注意：为避免任何差异，研究人员应立即解剖每个晶状体上皮囊并将它们暂时储存在 DPBS 中。只有在完成所有解剖后，胶囊才能集体转移到保持在37°C的胰蛋白酶中，确保同步和均匀的暴露。</li>
	<li>小心地将镜头囊转移到 6 孔板上。向每个孔中加入 1 mL 0.05% 胰蛋白酶溶液以启动酶解过程。</li>
	<li>轻轻搅拌胰蛋白酶溶液以确保均匀渗透。将板置于细胞培养箱中，让胶囊在37°C下消化8-10分钟。 注意：此步骤有助于晶状体囊组织的分解和随后单个上皮细胞的释放。</li>
	<li>孵育后，使用解剖剪刀小心地切碎消化的晶状体胶囊，以分解任何残留的组织团块并促进细胞分离。 注意：强调组织切碎的彻底性，以确保从消化的晶状体胶囊中有效释放细胞。</li>
	<li>加入 0.5 mL 含有 10% FBS 的培养基以淬灭胰蛋白酶。将组织样品转移到离心管中，并以1,000× g 离心5分钟。</li>
	<li>小心地除去上清液，不要干扰细胞沉淀。使用 1 mL 培养基重悬细胞并将细胞接种在 24 孔板中。</li>
	<li>每 2-3 天更换一次培养基。</li>
</ol>3. LECs亚文化
<ol><li>一旦细胞达到汇合，从培养皿中取出培养基。继续用 1 mL DPBS 洗涤细胞 2 次。</li>
	<li>加入 200 μL 胰蛋白酶-EDTA 溶液，将细胞置于培养箱中 5 分钟。</li>
	<li>孵育后，将细胞从培养箱中取出并在显微镜下检查它们，以确认它们已从培养皿中分离并开始漂浮。</li>
	<li>加入 1 mL 培养基，轻轻移液细胞 3-5 倍以分离所有细胞。</li>
	<li>将细胞转移到离心管中，并以1,000× g 离心5分钟。</li>
	<li>小心地除去上清液并将细胞重悬于完整的生长培养基中。</li>
	<li>如果需要，使用血细胞计数器计数细胞数量。</li>
	<li>以 1：2 或 1：3 的比例细分细胞悬液以进行传代培养。</li>
	<li>当培养物再次汇合时，重复上述过程。 注意：LEC在高密度培养条件下茁壮成长。避免过度稀释细胞，因为这可能会阻碍它们的生长。</li>
</ol>4.储存和运输
注意：理想的存储单元数是 ~1 × 106。
<ol><li>用 1 mL DPBS 彻底洗涤细胞 3 次。洗涤后，加入 1 mL 胰蛋白酶-EDTA 溶液，并将细胞置于培养箱中 5 分钟。</li>
	<li>加入 2 mL 完全培养基，将细胞悬液转移到离心管中，并以 1,000 × g 离心 5 分钟。</li>
	<li>弃去上清液并将细胞重悬于由 90% FBS 和 10% DMSO 组成的冷冻培养基中，目标是细胞密度为 1 ×10 6 个细胞/mL。将细胞悬液转移到冷冻管中。</li>
	<li>立即将细胞移至-20°C环境中1小时，然后在-80°C下过夜，然后永久储存在液氮中。 注意：如果没有液氮，则可以在-80°C下初始一小时后将细胞储存在-20°C。</li>
	<li>如果需要发货，请将冷冻管中的细胞装在装有干冰的包装中，以便隔夜交付。</li>
	<li>收到细胞后，确保快速回收并将细胞置于传代培养中。如果立即培养不可行，则将细胞转移到液氮中进行长时间储存。 注意： 如果要运送细胞，请确保样品深埋在干冰中，以防止温度波动造成潜在损坏。</li>
</ol>5. LEC验证
<ol><li>将LEC板入带有盖玻璃的35mm培养皿中，并培养约48小时。</li>
	<li>用PBS洗涤细胞2次，并在-20°C下用冷甲醇固定细胞10分钟。</li>
	<li>用PBS洗涤固定细胞3×5分钟，并在室温下用封闭缓冲液孵育固定细胞1小时，以防止非特异性结合。</li>
	<li>封闭后，将细胞在4°C下与一抗（αA-晶状体蛋白，γ-晶状体蛋白和PROX1抗体）在稀释缓冲液中以1：50的比例单独稀释。</li>
	<li>用PBS洗涤细胞3×5分钟，并将二抗在稀释缓冲液中以1：100稀释的二抗孵育1小时。</li>
	<li>用PBS洗涤细胞3×5分钟，并在室温下用PBS中的5μg/ mLHoechst 33342染色细胞10分钟以观察细胞核。</li>
	<li>用PBS洗涤细胞2×5分钟以除去多余的染色溶液，并使用荧光显微镜捕获细胞的荧光图像，使用DAPI通道用于细胞核，FITC通道用于αA-，γ-晶状体蛋白和PROX1。</li>
</ol>

晶状体上皮细胞的培养和表征

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本文介绍的方案为成功分离、培养和传代 LEC 提供了全面的分步指南，并附有视频文档。详细的视觉指南和书面说明增强了协议的清晰度和可访问性，促进了该领域研究人员的使用和可重复性。最终目的是促进围绕 LEC 在白内障形成和 PCO（白内障手术后普遍存在的并发症）中的作用的知识体系的扩展。
当将原代 LEC 与晶状体上皮细胞系（如 HLE-B3 和 SRA01/04）进行比较时，每种?...

眼睛的晶状体通过将入射光聚焦到视网膜上，在视觉中起着至关重要的作用。它由透明的无血管结构组成，由特化细胞组成，其中晶状体上皮细胞 （LEC） 是关键参与者。LEC 位于晶状体的前表面，负责保持其透明度、调节水平衡并参与晶状体生长和发育 1,2。LEC 是位于晶状体前部的一种独特类型的细胞，通过在整个生命周期中持续产生晶状体纤维，在维持晶状体清晰度和功能方面发挥着关键作用。
白内障的特征是晶状体逐渐混浊，导致光线失真和散射，导致视力受损 3,4。白内障形成的确切机制是复杂和多因素的，涉及各种细胞和分子过程，如紫外线辐射、氧化损伤和糖基化 5,6。已发现 LEC 对白内障的发展有显着贡献，使其成为研究的重要焦点 1,2,7,8,9。
此外，当今眼科最紧迫的问题之一是后囊混浊 （PCO） 的发生率相对较高，也称为继发性白内障。PCO 仍然是白内障手术后最常见的并发症，在手术后 5 年内影响多达 20-40% 的成人患者和 100% 的儿童10.PCO主要是由白内障摘除术后残留在囊袋中的LEC引起的。这些细胞经历多方面的病理生理学转化，不仅涉及上皮-间充质转化 （EMT），还涉及 LEC 向晶状体纤维的分化，从而形成 LEC、纤维和肌成纤维细胞的混合物细胞群 11,12,13。转化的细胞增殖并迁移到晶状体后囊，导致视力障碍。了解培养模型中 LEC 的行为和控制机制可以为 PCO 的预防和管理提供有价值的见解。因此，这种培养 LEC 的方案为眼科研究人员提供了一个重要的工具，旨在研究、理解并最终对抗这种普遍的术后并发症。
为了揭示LEC生物学的复杂性及其在白内障形成和PCO中的作用，必须建立强大且可重复的 体外 原代细胞培养系统。原代 LEC 培养为研究人员提供了一个受控环境来研究 LEC 的功能、信号传导和分子特征。此外，它还允许研究细胞过程和不同实验条件的影响，为晶状体生理学和病理学提供有价值的见解。
先前的研究丰富了我们对LEC培养技术的理解14,15,16,17,18,19,20。尽管这些研究采用了各种方法，并在LEC行为和特征方面取得了重要发现，但目前的文献中缺乏用于培养LEC的全面且可访问的视频记录方案。这种局限性会阻碍新手研究人员准确重现技术的能力，并可能导致实验结果的不一致和变化。通过提供视频录制协议，本文旨在弥合这一差距，并提供一种标准化资源，以提高LEC文化领域的可重复性并促进知识转移。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Thermo Fisher</td>
			<td>#25300054</td>
			<td>For LECs dissociation</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Secondary Antibody&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>#715-545-150</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-605-003</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Antibody dilution buffer</td>
			<td>Licor</td>
			<td>#927-60001</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Beaver safety knife</td>
			<td>Beaver-Visitec International</td>
			<td>#3782235</td>
			<td>For lens dissection</td>
		</tr>
		<tr>
			<td>Blocking buffer</td>
			<td>Licor</td>
			<td>#927-60001</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Capsulorhexis forceps</td>
			<td>Titan Medical Instruments</td>
			<td>TMF-124</td>
			<td>For lens capsule isolation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6429</td>
			<td>For LECs culture medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>#D2650</td>
			<td>For making freezing medium&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline&#160;</td>
			<td>Thermo Fisher</td>
			<td>#J67802</td>
			<td>For lens dissection</td>
		</tr>
		<tr>
			<td>Dumont tweezers</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-4976</td>
			<td>For lens capsule isolation</td>
		</tr>
		<tr>
			<td>EpiCGS-a (optional)</td>
			<td>ScienCell</td>
			<td>4182</td>
			<td>For LECs culture medium</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td>For LECs culture medium</td>
		</tr>
		<tr>
			<td>Gentamicin (50&#160;mg/mL)</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>For LECs culture medium</td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution</td>
			<td>Thermo Fisher</td>
			<td>#62249</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Roboz Surgical Instrument&#160;</td>
			<td>RS-5983</td>
			<td>For lens dissection</td>
		</tr>
		<tr>
			<td>Micro-dissecting tweezers</td>
			<td>Roboz Surgical Instrument&#160;</td>
			<td>RS5137&#160;</td>
			<td>For lens dissection</td>
		</tr>
		<tr>
			<td>PROX1 antibody</td>
			<td>Thermo Fisher</td>
			<td>11067-2-AP</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Vannas micro-dissecting spring scissors</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5608</td>
			<td>For lens capsule isolation</td>
		</tr>
		<tr>
			<td>&#945;A-crystallin antibody</td>
			<td>Santa Cruz</td>
			<td>sc-28306</td>
			<td>For cell validation&#160;</td>
		</tr>
		<tr>
			<td>&#947;-crystallin antibody</td>
			<td>Santa Cruz</td>
			<td>sc-365256</td>
			<td>For cell validation</td>
		</tr>
	</tbody>
</table>

optimizing mouse primary lens epithelial cell culture: a comprehensive guide to trypsinization

晶状体上皮细胞（LECs）在维持晶状体的稳态和正常功能方面发挥着多种重要作用。LEC 决定了晶状体的生长、发育、尺寸和透明度。相反，功能失调的 LEC 可导致白内障形成和后囊混浊 （PCO）。因此，建立强大的原代LEC培养系统对于从事晶状体开发、生物化学、白内障治疗和PCO预防的研究人员非常重要。然而，由于原代LECs的可用性有限、增殖速度慢且性质脆弱，其培育长期以来一直面临挑战。 
本研究通过提出原代 LEC 培养的综合方案来解决这些障碍。该方案包含基本步骤，例如配制优化的培养基、晶状体胶囊的精确分离、胰蛋白酶消化技术、传代培养程序、收获方案以及储存和运输指南。在整个培养过程中，使用相差显微镜监测细胞形态。 
为了确认培养的LEC的真实性，进行了免疫荧光测定以检测关键晶状体蛋白（即αA和γ晶状体蛋白）的存在和亚细胞分布。这一详细的方案为研究人员提供了培养和表征原发性 LEC 的宝贵资源，从而促进了我们对晶状体生物学的理解和晶状体相关疾病治疗策略的开发。

如 图2所示，通过遵循该方案，来自C57BL / 6J小鼠的原代LEC在4小时内粘附在培养皿上。值得注意的是，有其他组织的可见残留物，例如后囊和晶状体纤维细胞的部分。然而，这些意想不到的元素并没有附着在培养皿上，因此可以通过改变培养基来去除。随后，在第三天和第五天之间，LEC开始了其扩散阶段。然后，在第七天和第十天之间可以观察到快速增长，这是对数增殖阶段...

作者聚焦：揭开白内障的秘密 – 研究晶状体上皮细胞中的氧化还原修复酶

本手稿概述了用于培养原晶状体上皮细胞 （LEC） 的详细视频方案，旨在提高可重复性并帮助白内障和后囊混浊 （PCO） 的研究。它提供了有关晶状体解剖、LEC 分离和验证的分步说明，可作为有价值的指南，尤其是对于该领域的新手而言。

优化小鼠原晶状体上皮细胞培养：胰蛋白酶消化综合指南

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, ...

我们的研究目标是探索白内障的根本原因，特别强调氧化还原修复酶的功能。我们的目标是确定这种酶在晶状体保护中发挥的关键作用，并确定它们作为白内障预防和治疗药物靶点的潜力。在许多令人兴奋的技术中，我们的研究使用晶状体上皮细胞作为基础平台。这种方法对于揭示白内障形成、后囊混浊和发现新的抗白内障药物背后的机制至关重要。展望未来，我们将专注于了解抗氧化酶和衰老对镜片功能的影响。我们的目标是发现创新的抗氧化疗法，可以延缓晶状体老化，预防白内障，并为PCO提供有效的干预。

optimizing-mouse-primary-lens-epithelial-cell-culture-comprehensive

Research

JoVE Journal

Biology

Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization

1.7K 次观看.  University of North Texas Health Science Center. 我们的研究目标是探索白内障的根本原因，特别强调氧化还原修复酶的功能。我们的目标是确定这种酶在晶状体保护中发挥的关键作用，并确定它们作为白内障预防和治疗药物靶点的潜力。在许多令人兴奋的技术中，我们的研究使用晶状体上皮细胞作为基础平台。这种方法对于揭示白内障形成、后囊混浊和发现新的抗白内障药物背后的机制至关重要。展望未来，我们将?...

视频: 作者聚焦：揭开白内障的秘密 – 研究晶状体上皮细胞中的氧化还原修复酶

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

大鼠晶状体上皮细胞外植体的制备及文化学习终末分化

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

科研

生物学

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

实用指南系统发育的非专家

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

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 ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

盖玻片序贯应用评估鼠标透镜的抗压刚度:应变和形态分析

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

优化慢病毒生产和细胞培养必要条件，成功转导初级人支气管上皮细胞

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...