Name: induction and testing of hypoxia in cell culture
Uploaded: 2011-08-12T13:00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

1。缺氧诱导CoCl 2溶液钴（II）氯化（CoCl 2•6H 2 O，分子量= 237.9）是一种化学诱导缺氧诱导因子- 1.3 8。本产品易溶于水（100毫克/毫升），产生一个明确的，红色的解决方案。 <ol><li>准备一个25mm的库存解决方案，在无菌日水（准备使用前） </li><li>在常规的细胞培养介质使用CoCl 2诱导缺氧，在终浓度为100μm。 </li><li>添加CoCl 2包含媒体对你的细胞，并在传统的孵化器孵化24小时的文化（37 ° C，5％C0 2） 。 </li></ol>以上浓度的细胞，我们有测试，但应该是每一个细胞系在不同浓度（建立以剂量依赖曲线）测试以及在不同的孵育时间，以限制药物相关的毒性和优化检测线工程。 2。缺氧诱导模块化孵化厅<ol><li>准备至少有两个相同的（双胞胎文化）的细胞培养。可以在细胞培养瓶，板或培养皿。要打开总商会，首先打开位于室（用于注射吹扫缺氧气/管），轻轻地打开钢圈钳管上的两个白色塑料夹具。 </li><li>松开夹紧。现在可将盖子和托盘。 </li><li>将细胞培养在缺氧室。同时置于培养皿中，包含在会议厅内的无菌水，以提供足够的文化加湿。 </li><li>放置在常氧的“双胞胎”细胞培养作为对照。 </li><li>确保您的食品是安全的和不动，然后关闭盒盖室。正确定位钢圈夹，以确保密闭的腔封闭，并关闭它。 </li><li>要创建缺氧，管“缺氧罐”，含有1％O 2的混合气体。如果你有一个连接到你的坦克流量计，你的商会将直接连接到它（煤气罐流量计室）。我们使用在我们的稳压器中的流量计。 </li><li>重要的是去除大部分并非所有在您的媒体室和氧气目前，这样做，打开煤气罐在7-10分钟每分钟20公升的流量冲洗室;然后迅速关闭气体流量，并完全关闭，关闭两个白色夹子的室。 </li><li>所需的时间内返回会议厅，一个传统的孵化器。如果使用大型的文化，允许在媒体文化DE气体1 - 2小时，然后重复冲洗。 上述步骤的基础上构造函数的建议“（比卢普斯罗森堡，INC）。确保你煤气罐在所有的时间获得正确。 </li></ol>注： <ul><li>为了消除媒体中的O 2，建议将重瓦斯后1 -3小时室一次。 </li><li>孵化时间较长比48小时它也应重新气体室。 </li><li>氧传感器（电极/压力计），允许在细胞/室中的O 2的测量，将提供一个细胞内/室O 2的含）更精确的测量。 </li><li>培养细胞在不同的时间长度，时间曲线，将有助于确定的最佳时间在您的特定细胞中HIF -1α表达。 </li></ul> 3。缺氧的评价<ol><li> HIF - 1的免疫印迹检测α。 <ol><li>重新打开你的商会在第2.2节所述，立即置于冰上文化。 </li><li>然后用5％SDS在解决板块裂解缺氧治疗组和非治疗细胞转移细胞lyzate管，超声，转速减速（spindown）的细胞碎片，收集上清。 </li><li>测量蛋白质浓度用BCA试剂盒，准备样品加入样缓冲液，在95℃加热5分钟。 </li><li> Electrophorese 8％SDS聚丙烯酰胺凝胶转移到硝酸纤维素膜蛋白质。 </li><li>膜在PBS中，含有5％非脱脂奶粉和0.1％吐温20座在室温下1小时或4℃摇床过夜。 </li><li>免疫印迹法与反HIF -1α的主要单克隆抗体在同一个缓冲区（1 / 600）在4 ° C过夜</li><li>洗净，用含0.1％吐温20在室温下，5分钟，每次孵育HRP二级抗体（1 / 2000）的PBS + 0.1％吐温20为45分钟的3倍。 </li><li>洗净，用含0.1％吐温20在室温下，5分钟/时间的3倍。 </li><li>皮尔斯ECL试剂盒和X光片显示蛋白带</li></ol></li><li> 缺氧反应元件（HRE）。 HIF -1α调控许多基因（即血管内皮生长因子，促红细胞生成素），通过结合到一个序列称为缺氧调节元件（HRE）。使用人权教育相关的一个标记基因的我t是可以检测HIF活动9。若要使用此方法，首先你需要你的细胞转染与人权教育的荧光素酶质粒使用适合您的细胞转染方法的。例如，我们用脂质体2000。细胞需要转染至少4个小时之前，在低氧和常氧孵育。时间允许的DNA进入细胞，因此，这第一步不是缺氧的影响。使用荧光素酶检测试剂盒，荧光素酶检测到信号。 <ol><li>孵育您的HRE转染的细胞在缺氧模块化室或使用的HRE转染的细胞孵育CoCl 2，包括常氧控制。 </li><li>所需的潜伏期后，从细胞中移除的生长介质。 </li><li>在PBS冲洗细胞，去除PBS。 </li><li>免除到培养皿400μl1X裂解试剂，并刮去贴壁细胞，然后将它们传输到一个微型管。 </li><li>短暂离心颗粒碎片。 </li><li>混合20μL细胞裂解液上清与萤光素酶检测试剂100μL和使用光度计测量发光。 </li></ol></li></ol> 4。代表性的成果： 在K562细胞（人白血病细胞株）培养2天，含氧量低，相比常氧，缺氧诱导增加HIF -1α蛋白免疫印迹（图1）检测。 使用的HRE -荧光素酶改性293细胞（人胚肾细胞系）在缺氧培养，显着增加，在缺氧细胞HIF -1α活动检测（图2）。 <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="图1"/> 图1：增加缺氧细胞HIF -1α。 K562细胞在常氧和缺氧（缺氧室）培养48小时，并使用一种抗体具体为HIF -1α的西部印迹分析。为肌动蛋白抗体的具体用于装载控制。 <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="图2"/> 图2：HIF -1α的活动 。荧光素酶的HRE修改293细胞培养在低氧和常氧48小时，然后裂解使用荧光素酶检测试剂盒和光度计检测荧光素酶的信号。结果表示为相对发光单位（RLU）。

<ol>
	<li>Swinson, D. E., O'Byrne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+hypoxia+and+epidermal+growth+factor+receptor+in+non-small-cell+lung+cancer.">Interactions between hypoxia and epidermal growth factor receptor in non-small-cell lung cancer.</a> Clin Lung Cancer. 7, 250-256 (2006).</li><li>van Laarhoven, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+in+relation+to+vasculature+and+proliferation+in+liver+metastases+in+patients+with+colorectal+cancer.">Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer.</a> Int J Radiat Oncol Biol Phys. 64, 473-482 (2006).</li><li>Hockel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+pO2+predicts+survival+in+advanced+cancer+of+the+uterine+cervix.">Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix.</a> Radiother Oncol. 26, 45-50 (1993).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1+and+human+disease:+one+highly+involved+factor.">HIF-1 and human disease: one highly involved factor.</a> Genes Dev. 14, 1983-1991 (2000).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1:+master+regulator+of+O2+homeostasis.">Hypoxia-inducible factor 1: master regulator of O2 homeostasis.</a> Curr Opin Genet Dev. 8, 588-594 (1998).</li><li>Tatum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82, 699-757 (2006).</li><li>Brahimi-Horn, M. C., Pouyssegur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF+at+a+glance.">HIF at a glance.</a> J Cell Sci. 122, 1055-1057 (2009).</li><li>Piret, J. P., Mottet, D., Raes, M., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CoCl2,+a+chemical+inducer+of+hypoxia-inducible+factor-1,+and+hypoxia+reduce+apoptotic+cell+death+in+hepatoma+cell+line+HepG2.">CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2.</a> Ann N Y Acad Sci. 973, 443-447 (2002).</li><li>Emerling, B. M., Weinberg, F., Liu, J. L., Mak, T. W., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+regulates+p300-dependent+hypoxia-inducible+factor+1+transcriptional+activity+through+Forkhead+transcription+factor+3a+(FOXO3a).">PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a).</a> Proc Natl Acad Sci U S A. 105, 2622-2627 (2008).</li></ol>

没有利益冲突的声明。

细胞增殖和缺氧条件下的生存能力有很大的差异，根据细胞类型。因此，你应该调整细胞数或数量，以确保你将有足够的细胞/蛋白质为您的实验开始的文化板块。 氯化钴方法的优点是价格低廉和快速。本产品模仿缺氧诱导HIF-1/3α，但还可以调节其他基因，确保检查什么其他的影响，它可能对您的特定细胞的功能和表型的“模仿独立本产品适合您的项目缺氧“的效果。另一种药物也可以用来模仿缺氧是去铁胺甲磺酸（DFO，终浓度为100微米）。这些药物的使用，允许experimentator开放，而不影响“缺氧”的培养板/盘/瓶多次。 混合气体中的氧气水平可以有所不同，取决于您的实验和细胞类型，缺氧值的变化对组织和细胞类型的不同而不同。事实上，一些细胞缺氧5％O 2的其他需要不到1％O 2的缺氧。添加5％的CO 2混合气体，以稳定的文化的pH值，其余的气体通常是氮气。 缺氧商会有没有使用药物可以替代细胞行为的独立的氧张力的优势。然而，并非所有类型的实验可以做氧气重新进入在每个开放的腔，从而减轻缺氧。你应该考虑这个因素在您的实验;缺氧/复氧是一种特定条件下，可能会影响一些类型的细胞。另一种方法是使用缺氧工作站（预混合气体罐或气体混合系统连接到）或较大缺氧培养箱（缺氧处理室，手套箱），允许试验，以改变媒体和操纵连续缺氧环境中的细胞。各种缺氧商会在过去的十年，已经商业化的选择应根据您的实验室使用，项目的空间，大小，和预算。一定要确保用好你的房间和条件，以保证正确的文化条件。一般情况下，用一腔时，缺氧的水平可以通过选择不同的气体混合物（即1％，5％〜10％的氧气）的调制。 关于中HIF -1α的检测，重要的是要知道，某些肿瘤细胞系表达常氧HIF -1α。因此，使用常氧控制，以确定这些细胞系中HIF基础水平的关键。 HIF -1α也可以在非恶性细胞常氧细胞刺激或压力。这也可能发生，如果这些细胞被饿死，确保您的细胞培养正确的饲料和保持。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>反HIF -1α </td><td> Invitrogen公司</td><td> 458400 </td></tr><tr><td> （二）氯化钴</td><td>西格玛</td><td> C8661 - 25G </td></tr><tr><td>孵化商会</td><td>比卢普斯，罗滕伯格</td><td> MIC - 101 </td></tr><tr><td>的HRE荧光素酶质粒</td><td> Adgene </td><td>质粒26731 </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

缺氧是指器官，组织或细胞减少或缺乏氧气。这可能是由于在氧气的供应量减少（的原因包括没有足够的血管网络，有缺陷的血管，和贫血）或增加氧气供应（突然更高的细胞增殖率造成消费相对的氧张力下降） 。缺氧可，如在固体癌症 1-3，类风湿关节炎，动脉粥样硬化等病理生理或... ...每一个组织和细胞具有不同的能力，以适应这一新的条件。在缺氧，缺氧诱导因子（HIF）是稳定和调节不同的基因，如参与血管生成或运输氧气 4的。这种蛋白的稳定是一个缺氧的标志，因此，检测HIF是经常使用到屏幕缺氧5-7上。 在这篇文章中，我们提出两个简单的方法，缺氧诱导哺乳动物细胞培养和简单的测试，以评估这些细胞的缺氧状态。

Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

Induction and Testing of Hypoxia in Cell Culture

在这里，我们提出简单的方法，诱导缺氧细胞培养和简单的测试，以评估文化的缺氧状态。

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 ...

induction-and-testing-of-hypoxia-in-cell-culture

Research

JoVE Journal

Biology

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

培养小鼠神经干细胞的前体

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

科研

生物学

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

收养大鼠迟发型超敏反应的感应和监测

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

在c-fos蛋白免疫组织学检测：一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.