晶状体上皮植的准备工作已经改编自起源于实验室的博士约翰麦卡沃伊9的方法。这项工作是由美国国家眼科研究所，院内研究计划Z01 - EY000238 - 22

第1部分：去除镜片材料与试剂： <ol><li>微解剖剪刀，弯曲，生硬的提示，（如as.RS - 5983，Roboz手术器械有限责任公司，马里兰州盖瑟斯堡）;微解剖镊子，弯尖（如Roboz＃RS5137）。 </li><li>悬浮介质：中等199含0.1％牛血清白蛋白，100单位/ ml青霉素，100μg/ml链霉素，2.5微克/毫升两性霉素B试剂可以从Invitrogen公司，卡尔斯巴德，CA。 </li></ol>程序： <ol><li>安乐死新生大鼠（年龄在2-4天），按照卫生，马里兰州贝塞斯达的国立研究所提供的指引。 </li><li>卸下眼睑手术剪刀。眼圈两侧弯曲镊子轻轻按下，迫使眼睛向外凸出。后路侧眼用剪刀做一个小切口。按下镊子与反对方眼对面的切口，然后被强迫的镜头和一个附加玻璃体少量通过破裂，使镜头被拾起弯曲镊子。应采取不打破晶状体囊。 </li><li>使用弯曲的镊子镜片转移到60毫米塑料组织培养皿中含有5毫升温暖，无菌的悬浮介质。 </li></ol>第2部分：外植体的显微切割材料与试剂： <ol><li>微解剖镊子，0.1毫米的提示，（如RS - 4976 Roboz）。我们使用，因为尖端的灵活性Dumostar合金，但也是可以接受的其他合金钢的。提示厚度小于0.1mm的镊子是不建议，因为他们往往​​在此过程中折断或弯曲。 </li><li>悬浮介质（见第1部分） </li><li> Unsupplemented火腿的F12中等或中等199（Invitrogen公司，Carlsburg，CA） </li></ol>程序： <ol><li>应进行下列步骤，在一个干净的草案，免费使用无菌解剖工具和无菌介质环境。 </li><li>使用立体解剖显微镜，任何坚持组织镜片清洁与0.1毫米尖镊子，将它们传输到第二个60毫米培养皿中含有5毫升无菌悬浮介质（这一步，有利于减少污染）。用镊子，将所需数量的镜头，一个35mm的培养皿中含有5毫升无菌，unsupplemented中等。 （我们经常使用这一步火腿的F12（Invitrogen公司），因为在较低的染料浓度使清扫容易，但是，中等199是还可以接受任何抗生素或其它添加内容是在这一步需要。。）由于许多为12外植体可以被制成在一盘这种规模的中心。然而，一个35毫米的菜应使用，即使少植，解剖工具，因为不能在一个较小的菜很容易被操纵。转让的菜，含有其余的镜头一个组织文化的孵化器，在37 ° C直到他们需要的。 </li><li>确定的镜头后的一面。认识到这一点的方法有很多：1）后比前，这是略扁平的圆; 2）新生鼠镜头形成冷白内障，因为他们凉在清扫室温。如果冷白内障还没有完全填补镜头，后一边是从不透明区域最远的一侧。如果不透明度，填补了镜头，气候变暖的菜到37 ° C的几分钟内将扭转这种局面。后侧面可以被认定为冷却后的白内障改革。 3） 白膜vasculosa晶状体的痕迹，可能后可见。 4）后缝合后可见。正确识别后一边是必不可少的，因为这是将胶囊打开。开幕式上的镜头前侧撕裂的上皮细胞。 </li><li>一旦后已经确定，把它向上和把握在左镊子镜头（右手工人）。然后捏住镊子产生一个小的折叠后囊。 </li><li>按住后囊，把握正确的镊子左镊子胶囊倍，拉两对镊子，在相反的方向，使小囊中撕裂。 </li><li>在抓镊子缩回胶囊的边缘，将它拉向下，首先在一边，然后，另一方面，它压入菜用的镊子的塑料。重复了几次，围绕赤道的镜头移动，直到胶囊牢固地附着在板材多点。在左镊子胶囊，轻轻摇动纤维质量与正确的镊子，打破附着在晶状体赤道之间的纤维细胞和上皮细胞。然后推动纤维质量，滚动胶囊/上皮细胞，这仍然是底部菜。继续这个过程中，在盘子里的所有镜头。删除和丢弃的纤维群众（或将它们保存为其他研究的晶状体蛋白源冻结）。 </li></ol>第3部分：中央外植体的显微切割和文化材料与试剂： <ol><li>一次性无菌手术刀，15号刀片，（手术有限公司辛辛那提，俄亥俄州辛辛那提） </li><li>无菌磷酸盐缓冲生理盐水与钙和镁（Invitrogen公司，卡尔斯巴德，加利福尼亚州） </li><li>培养基：含100ng/ml FGF - 2（Sigma - Aldrich公司，圣路易斯，密苏里州）的悬浮介质。 </li></ol>程序： <ol><li>用消毒的手术刀，修剪掉周边上皮（PE），其中包含细胞分化的早期阶段（图1A），离开中央广场约2.0毫米，中央上皮细胞组成的一个侧面，（CE认证）（图1B）。 </li><li>传输的中央植菜含有一个生物安全柜。无菌PBS含有钙和镁的一次新鲜，平衡（37 ° C，5％CO 2）悬浮介质1毫升清洗3次。这些清洗污染的可能性大大降低。 </li><li>添加培养基2ML，诱导分化 1，并将其放置在饱和湿度，组织培养孵化器的外植体在37 ° C，5％的CO 2。文化时期可能只要短短几个小时，或延长，只要两至三个星期，根据正在研究的参数。中期应改为每两至三天。 </li></ol>第4部分：外植体的收获与分化相关的事件的分析 1。分析蛋白质或RNA的收获植材料： <ol><li> Microdissecting镊子</li><li> SDS裂解液，或RNA后</li></ol>程序： <ol><li>在潜伏期末，去除培养基。 </li><li>使用立体解剖显微镜，轻轻地松开每个外植体的边缘。 </li><li>用镊子提起每个外植体，并将其转移到100μLSDS裂解液（蛋白质分析）或RNA后（RNA分析）包含一管。刺激的解决方案中的镊子尖端，以确保外植体不沾镊子。 </li></ol> 2。免疫收获植材料与试剂： <ol><li>磷酸盐缓冲生理盐水与钙和镁（Invitrogen公司，卡尔斯巴德，加利福尼亚州） </li><li>磷酸盐缓冲液（无钙和镁的）（Invitrogen公司，卡尔斯巴德，CA） </li><li> 4％多聚甲醛（波士顿生物制品，马萨诸塞州伍斯特市） </li><li> Microdissecting镊子</li><li>疏水标记笔</li><li>显微镜幻灯片</li><li> 0.25％的Triton X - 100的磷酸盐缓冲液。 </li></ol>程序： <ol><li> PBS中含有钙，镁等，简单地冲洗植。 </li><li>加入4％多聚甲醛在室温下30分钟，修复组织。 </li><li>取出固定液，用磷酸盐缓冲液取代。固定植变得有点僵硬，让他们解除和免疫转移到玻片。 </li><li>将幻灯片上的位置，以帮助组织和防止卷曲的小下降的PBS（不包括钙和镁的）。使用microdissecting镊子，电梯外植体，并把它插入到幻灯片上的下降。不接触外植体，小心地取出一张纸烛芯的液体外植体上的玻璃压平，让组织在空气干燥，在室温下3〜5分钟。 </li></ol><img src="/files/ftp_upload/1519/fig1.jpg" alt="图1" /> 图1 A的周边上皮细胞表达分化的特定蛋白质。显示外植体为N - cadherin的免疫染色后立即显微切割。表达被认为是在一个乐队在周边上皮细胞，表明细胞在这一地区已开始分化。B.，周边上皮可削去去除细胞表达N - cadherin的和其他分化的特定蛋白质。红色环代表表达N - cadherin的，而灰色概述小方代表象限面板1A所示上皮细胞的位置。四个手术刀切割周边上皮可去除，留下了中央广​​场，仅包含尚未开始分化的细胞。

<ol>
	<li>McAvoy, J. W., Chamberlain, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+(FGF)+induces+different+responses+in+lens+epithelial+cells+depending+on+its+concentration.">Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration.</a> Development. 107 (2), 221-228 (1989).</li><li>Campbell, M. T., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+of+fibre+differentiation+in+cultured+rat+lens+epithelium+under+the+influence+of+neural+retina-conditioned+medium.">Onset of fibre differentiation in cultured rat lens epithelium under the influence of neural retina-conditioned medium.</a> Exp Eye Res. 39 (1), 83-94 (1984).</li><li>Lovicu, F. J., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+lens+epithelial+explants+induced+to+differentiate+into+fibres+by+fibroblast+growth+factor+(FGF).">Structural analysis of lens epithelial explants induced to differentiate into fibres by fibroblast growth factor (FGF).</a> Exp Eye Res. 49 (3), 479-494 (1989).</li><li>Golestaneh, N., Fan, J., Fariss, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+major+intrinsic+protein+(MIP)/aquaporin+0+expression+in+rat+lens+epithelia+explants+requires+fibroblast+growth+factor-induced+ERK+and+JNK+signaling.">Lens major intrinsic protein (MIP)/aquaporin 0 expression in rat lens epithelia explants requires fibroblast growth factor-induced ERK and JNK signaling.</a> J Biol Chem. 279 (30), 31813-31822 (2004).</li><li>Saravanamuthu, S. S., Gao, C. Y., Zelenka, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+is+required+for+lateral+induction+of+Jagged1+during+FGF-induced+lens+fiber+differentiation.">Notch signaling is required for lateral induction of Jagged1 during FGF-induced lens fiber differentiation.</a> Dev Biol. 332 (1), 166-176 (2009).</li><li>Lovicu, F. J., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-induced+lens+cell+proliferation+and+differentiation+is+dependent+on+MAPK+(ERK1/2)+signalling.">FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling.</a> Development. 128 (24), 5075-5084 (2001).</li><li>Dirks, R. P., Kraft, H. J., Van Genesen, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cooperation+between+two+silencers+creates+an+enhancer+element+that+controls+both+the+lens-preferred+and+the+differentiation+stage-specific+expression+of+the+rat+beta+B2-crystallin+gene.">The cooperation between two silencers creates an enhancer element that controls both the lens-preferred and the differentiation stage-specific expression of the rat beta B2-crystallin gene.</a> Eur J Biochem. 239 (1), 23-32 (1996).</li><li>Yang, Y., Stopka, T., Golestaneh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+alphaA-crystallin+via+Pax6,+c-Maf,+CREB+and+a+broad+domain+of+lens-specific+chromatin.">Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin.</a> The EMBO journal. 25 (10), 2107-2118 (2006).</li><li>McAvoy, J. W., T, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+retinas+promote+cell+division+and+fibre+differentiation+in+lens+epithelial+explants.">Neural retinas promote cell division and fibre differentiation in lens epithelial explants.</a> Curr Eye Res. 3 (6), 827-834 (1984).</li></ol>

大鼠晶状体植体系统，已成功地用于由一个实验室，研究终端晶状体上皮细胞分化为晶状体纤维 21,3,4,5 。当暴露在100ng/ml FGF - 2的外植体将开始显示在几分钟之内6信号的变化，在形态和基因表达在 3,4,5几天连续出现的变化。如果护理是采取措施防止污染的文化仍然可行2-3周。 在本议定书中所描述的中央植学习分化相关的事件顺序特别有用，因为它们包含几个如果任何细胞表达FGF - 2之前的分化标记添加1,5。细胞分化同步，作为一个队列中，使其能够按照信令和分化相关的转录事件的时间当然。培养时间长短不同外植体，从而提供准确的时间信息的顺序事件。特异性抑制剂可添加到培养基中，以确定有关的信号通路。可用于植SDS凝胶电泳和免疫印迹分析蛋白表达或以特定的mRNA经RT - PCR分析的表达。蛋白质产量从20-50微克/外植体和RNA产量的范围大约是200 - 600毫微克/外植体，取决于文化时期的长度。我们一般5-6个菜植几个检测提供足够的蛋白质或RNA。从外植体的RNA也可以用来准备评估，通过基因芯片分析，它可以识别新基因，可能是分化的关键基因表达的cDNA。植也可以转。虽然转染效率普遍偏低，这是足够的化验报告基因4; 7; 8。免疫荧光显微镜外植体提供了一个有用的辅助生化方法确定利益的蛋白质的亚细胞定位。因此，编制和大鼠晶状体植的文化提供了一个功能强大的系统研究在哺乳动物终端镜头分化，可以补充体内的技术，如转基因和基因敲除小鼠的一代，的。

preparation and culture of rat lens epithelial explants for studying terminal differentiation

目镜的前表面覆盖由单层上皮细胞，在一个环形睫状体区底层增殖。司以下的，这些细胞迁移后方，FGF从视网膜扩散诱导它们分化成一个拉长的镜头纤维细胞，撰写大部分的镜头后的数组。晶状体上皮细胞分化成晶状体纤维可在体外诱导培养外植体的中部地区前上皮细胞中FGF - 2的存在。植准备，消除眼睛的镜头和把握晶状体囊解剖镊子后从新生大鼠镜头。后囊，然后轻轻地撕开，并压成一个组织培养皿塑料底。用手术刀将被删除外植体周边地区和中部地区，然后培养在FGF - 2 100ng/ml只要2-3周的存在，取决于要研究的参数。由于培养外植体的上皮细胞分化近似同步信号和基因表达的时间超过几天到几周内，当然可以决定使用分子，生化和药理技术。免疫荧光显微镜是一个功能强大的辅助手段，这些方法，因为它表明利益的蛋白质的亚细胞定位，并能够揭示信号转导通路的实验操作的生理后果。

Watch this Scientific Journal Video about Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation at JoVE.com

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

在FGF - 2的存在培养的大鼠晶状体上皮细胞的中部地区的外植体分化同步。这种文化免疫荧光显微镜，可以提供有关新的信息与终末分化相关的基因表达和信号事件。

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.  ...

preparation-culture-rat-lens-epithelial-explants-for-studying

Research

JoVE Journal

Biology

12.6K 次观看.  National Eye Institute (NEI), National Institutes of Health (NIH). 在FGF - 2的存在培养的大鼠晶状体上皮细胞的中部地区的外植体分化同步。这种文化免疫荧光显微镜，可以提供有关新的信息与终末分化相关的基因表达和信号事件。

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

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

器官型E18大鼠脑切片文化

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

科研

生物学

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

从外植体生长的首要人权支气管上皮细胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

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, ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

文化与偏振刺激人体肠道黏膜植的新方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

大鼠血清适用于哺乳动物全胚胎培养的制备

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

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 ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

大鼠骨髓破骨衣原体的分离、纯化及鉴别

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

研究鸡晶状体发育过程中的蛋白质表达

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

作者聚焦：通过在胚胎鸡晶状体中显微注射 RCAS（A） 逆转录病毒来研究晶状体的发育和功能 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

重组RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...