我们要感谢宽广的干细胞研究中心FACS，尤其要感谢他们的帮助杰西卡·斯科尔斯和Felicia科德雷亚与细胞分选。这项工作是由CIRM RN2-00904-1，K08 HL074229，美国胸科学会/慢性阻塞性肺病基金会ATS-06-065，值得关注的基金会，加州大学洛杉矶分校琼森综合癌症中心胸腔肿瘤计画/肺癌SPORE大学，加州癌症研究协调委员会和格温哈森樱桃纪念实验室（BG）。

纲要的步骤 1。解剖气管 2。清洁气管和切割 3。酶消化，加工成单细胞悬液 4。染色流式细胞仪和分选 5。排序细胞在体内和体外模型处理 1。解剖气管<ol><li>安乐死鼠标0.1 mg/0.2 cc的戊巴比妥钠腹膜内注射。 </li><li>剖开腹壁，移动侧的肠子暴露腹主动脉，然后对它进行解剖。 </li><li>打开胸部，通过双方的隔膜和前胸壁上切割，暴露心脏和肺部。继续到颈部去除脂肪和唾液腺暴露气管切割。 </li><li>取出胸腺和关闭连接下面和后面切肺。 SEPAR吃了从食管气管插入他们之间的钳子，然后在其上大部分高于其与喉切开气管。然后按住气管钳和分析及其附件，并继续向下与心脏和肺的块将其删除。包括大部分的左，右主支气管肺门附近的心脏和肺切掉。 </li><li>将含有在冰上收集介质在一个小的陪替氏培养皿气管。 </li></ol> 2。清洁气管和切割<ol><li>在解剖显微镜下，用极细的点钳（第5号）和“Vannas蒂宾根剪刀（FST 15003-08 finescience.com），清洁气管连接到它的所有其他组织。这包括：淋巴结肿大，喉返神经，脂肪，甲状腺，剩余的肺血管。气管的上半部分需要特别注意，以消除大喉软骨无影响最大的一堆竞彩提交之间的环状软骨和优先气管软骨环（C1）4。 </li><li>剪切气管进入C4及C5之间的上部和下部，然后切开气管的管腔通过两个零件。 </li></ol> 3。酶消化和加工成单细胞悬液（SCS） <ol><li>孵育气管的下部，在1毫升16 U分离酶在Eppendorf管中，在室温下（RT）30分钟后，如先前所述5。然后添加一个额外的20分钟，RT 0.5毫克/毫升的DNA酶I DMEM。 </li><li>取出消化气管新鲜的收集介质的无菌培养皿中，在解剖显微镜下剥离表面上皮。使用移液管转移的介质含有剥离的上皮50毫升锥形管。 </li><li>自旋向下剥离在1000 XG上皮细胞在4°C。取出上清液，并加入0.1％胰蛋​​白酶/ EDTA，并培育一阵晃动日Ê管中，在37℃下摇床（200rpm）下30分钟。培养后，使用1,000微升小费，吸取试剂和来打破团块和形成单细胞悬浮液。 </li><li>将含有1毫升0.15％链霉蛋白酶在锥形管中的上部气管。然后在4℃下保温4小时。 </li><li>振荡管，然后将气管进入新鲜收集介质，并确认在解剖显微镜下，是完全脱离表面上皮（SE）。 </li><li>气管剁碎，用细剪刀打开了SMG的车厢，然后在室温下培养一个多小时的蛋白酶缓慢的晃动。 </li><li>自旋向下的气管组织，消除蛋白酶，增加0.1％胰蛋​​白酶/ EDTA和孵化，在37°C，30分钟颤抖以上。 </li><li>通道的消化细胞，一丛丛，任何剩余的腺体块通过20G，23G和26G的针头，用10毫升的注射器。重复几次传代，每一个针头大小的前下台。现在，大多数细胞应该是在SCS。 </li><li>通过一个40微米的过滤器过滤悬浮液，然后清洗过滤器和自旋向下的细胞。重组细胞在适当体积的介质，细胞计数，并立即着手FACS染色和排序。不要让细胞在4℃或冰上几个小时，因为我们发现，这种影响的可行性和严重降低了球体形成效率。 </li></ol> 4。染色流式细胞仪和排序<ol><li>以足够的细胞用于制备未染色的和单一的彩色补偿管，然后染色，剩下的细胞与初级抗体，山羊的TROP2和大鼠ITGA6，在RT下的15分钟。然后用PBS或收集介质，去除上清液非常小心，打破了细胞沉淀。 </li><li>色斑与适当的辅助抗体在室温下10分钟，在黑暗中孵化，洗净，除去上清液，再破颗粒。 </li><li>在FACSARIA，调整后的电压和补偿，门出来的碎片，非常小的细胞，细胞的双峰和死亡的细胞。然后，大部分细胞从SE应该是TROP2阳性，而20％至50％的SMG细胞TROP2 + 6。 </li><li>设置您的选门TROP2 + ITGA6 +细胞从SE（基底细胞）和TROP2 +细胞（+ /-ITGA6阳性细胞）由SMG（管细胞）。 </li></ol> 5。加工分选细胞， 在体外培养和体内模型 5.1 在体外球体形成5-9文化<ol><li>混合100μl的“生长因子减少Matrigel的”用100μl的完全培养基10,11（ 见表1），含有多达50000排序细胞，然后把该混合物中的上部腔室24孔的反式阱。然后将400μl的完全培养基中，在下部腔室中孵育在37℃下改变介质在下部腔室隔日和添加额外的100微升的Matrigel每一周或更早的版本，如果它变得薄。 </li></ol> 5.2 体内模型<ol><li>重新构成的排序条件细胞沉淀，在完全培养基1:1混合的5,000-10,000的细胞每50μl的浓度与基底膜。 </li><li>麻醉收件人C57/BL6小鼠。管理卡洛芬（5毫克/公斤）subcutaenously作为镇痛剂。消毒刮他们的背上，然后在上背部中央纵向切开皮肤。切口应足够大，以确定的脂肪垫，以及注射部位，注射后形成的滤过泡。使用的剪刀钝面扩大袋之间的皮肤和背部的两侧胸壁上最大限度地获取和可视化。 </li><li>通过按下鼠标的前肢和落后的肩胛骨移动确定的肩胛骨内侧缘。取50微升的细胞悬浮液，并将它注入到300微升的BD胰岛素注射器只是在脂肪垫内侧肩胛骨和横向椎骨。你应该能看到一个水泡，否则你太深。然后剪辑或缝合皮肤切口的另一侧，重复相同的。查看小鼠和得到卡洛芬（5毫克/公斤，南卡罗来纳州），每日一次，48小时。 </li><li> 3周后，安乐死与pentobarbatol（0.1 mg/0.2毫升，IP）的小鼠，打开上述的皮肤和剖析双方的安全余量，以避免错过任何可能形成的组织结构，并延伸至整个脂肪垫。修复PFA和嵌入在石蜡12。在修整和切割块，绕过或忽略的结构，应格外小心。最好的做法是修剪一次，然后切成4μm的切片，将其放置在载玻片上，在显微镜下检查它，以确认是否有任何“上皮结构”这一部分的块或40微米。如果没有看到，然后修剪一次，然后重复此步骤。 </li></ol> 6。代表性的成果 S饰鼠标气管的pping将导致在剥蚀气管内，如在图1A中所示，是通过光学显微镜可见， 图1B示出后的气管上皮剥离从组织和类似葡萄串被释放的粘膜下腺体的明视野图。 链霉蛋白酶删除的ITGA6的抗原表位，这是为什么的的TROP2管细胞可以不被分成ITGA6 +和 - 人口。然而，分散酶，其中保存着ITGA6表位，消化所有的表面上皮细胞即使有很短的孵育时间和管道，并因此不能被用于分离管从基底细胞3。流式细胞术的单细胞悬浮液应显示正散射和侧散点图，在图2A中表示的。的，如果气管细胞TROP2抗体可以看出，在流式细胞仪荧光激活细胞sorti的导管细胞的其余部分良好的分离纳克（FACS），如在图2B中看到。球体应该是可见的，在的基底膜在培养约1周后，在外观上是致密的，如在图3中示出。这个过程的顺序是1-2％，单细胞的效率将仍然存在于基底膜，可能代表细胞​​的粘膜下腺导管不具备自我更新的能力。导管干细胞注射到小鼠脂肪垫的查询结果粘膜下腺状结构， 如图4所示形成。这些块中，即使没有H＆E染色。这些是球形和具有中央管腔。为了不要错过这些上皮的结构，需要做许多的横截面，通过脂肪垫。 <img alt="图1" src="/files/ftp_upload/3731/3731fig1.jpg" /> 图1。气道上皮细胞的酶消化。去除表面上皮细胞示出与黑单肩休闲包K表箭头，切除粘膜下腺导管细胞显示绿色箭头。示出30分钟的分散酶消化后，链霉蛋白酶消化4小时后，用光学显微镜可见的剥蚀气管。 <img alt="图2A" src="/files/ftp_upload/3731/3731fig2A.jpg" /> 图2（A）。流式细胞仪代表前向散射和侧向散射光图从小鼠气管表面上皮剥离后的单细胞悬液。 （二）代表FACS图TROP2从小鼠气管导管细胞表达的。 <img alt="图2B" src="/files/ftp_upload/3731/3731fig2B.jpg" /> 图2B。流式细胞仪的单细胞悬液从精简的表面上皮的低三分之二的小鼠气管代表前向散射和侧向散射光图。 （二）典型的FACS情节ITGA6表达的苏rface上皮细胞。 III。另一位代表的的FACS情节的正向和侧向散射光从表面上皮门的绿色基底细胞人口。 （四）典型ITGA6 TROP2表达细胞亚群的绿色。 <img alt="图4" src="/files/ftp_upload/3731/3731fig3.jpg" /> 图3。发展领域的时间过程。 A.管道领域。球在基底膜约1周后的文化应该是可见的，几乎全是密集的外观。单细胞不形成球体也看到了。 （比例尺= 50微米）。 B.基底球是在外观上和管腔。单细胞不形成球体也看到了。 （比例尺= 50微米）。 <img alt="图4" src="/files/ftp_upload/3731/3731fig4.jpg" /> 图4。导管干细胞注射到小鼠脂肪垫的查询结果粘膜下腺状结构的形成中。代表结构显示中的脂肪垫和一些人产生粘蛋白（淡蓝色），阿尔辛蓝碘酸雪夫染色。

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没有利益冲突的声明。

这种技术来隔离气道管和基底细胞重要的是，提高我们对气道的修复和再生，气道疾病。这里描述的技术包括几个关键的步骤。第一是优化酶消化期间。第二个是创造的单细胞悬液通过串行逐步提高计针，以防止传代细胞剪切，但分手的细胞团块。第三个是FACS分析，并适当的抗体的细胞的门控。 由于一些酶带钢表面抗原表位，像ITGA6和NGFR，一个可能的修改是改变内切酶酶切?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td></tr><tr><td>完成媒体10： DMEM-F-12，50/50，1X） </td><td>敏达</td><td> 15-090-CV </td></tr><tr><td> HEPES（15毫米） </td><td> Invitrogen公司</td><td> 15630 </td></tr><tr><td>碳酸氢钠（3.6毫米或0.03％） </td><td> Invitrogen公司</td><td> 25080 </td></tr><tr><td> （4毫摩尔）L-谷氨酰胺</td><td>敏达</td><td> 25-005-CL </td></tr><tr><td>青霉素（100 U / ml的） </td><td>敏达</td><td> 30-001-CI </td></tr><tr><td>链霉素（100微克/立方米） </td><td>敏达</td><td> 30-001-CI </td></tr><tr><td> 两性霉素B（0.25微克/毫升） </td><td> 龙沙 </td><td> 17-836R </td></tr><tr><td>在胰岛素（10微克/毫升） </td><td>西格玛</td><td> I6634 </td></tr><tr><td>转铁蛋白（5微克/毫升） </td><td>西格玛</td><td> T1147 </td></tr><tr><td>霍乱毒素（0.1微克/毫升） </td><td>西格玛</td><td> C8052 </td></tr><tr><td>表皮生长因子（25毫微克/毫升） </td><td> BD </td><td> 354001 </td></tr><tr><td>牛垂体提取物（30微克/毫升） </td><td> Invitrogen公司</td><td> 13028-014 </td></tr><tr><td>胎牛血清（5％） </td><td>费舍尔</td><td> SH3008803HI </td></tr><tr><td>视黄酸（0.05微米） </td><td>西格玛</td><td> R2625 </td></tr><tr><td>生长因子减少基底膜</td><td> BD </td><td> 354230 </td></tr></tbody></table> 表1。完整的媒体组件。 <table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </TD&gt; <td> 目录编号 </td><td> 评论 </td></tr><tr><td>链霉蛋白酶</td><td>罗氏公司</td><td> 10165921001 </td><td>用于在0.15％： O / N在4°C消化，到提取总的气管细胞（ALI文化） -4小时消化4°C隔离SMG </td></tr><tr><td>中性蛋白酶</td><td> BD Biosciences公司</td><td> 354235 </td><td> 16单位：30分钟RT </td></tr><tr><td> DNA酶I </td><td>西格玛</td><td> DN25 </td><td>适用于0.5毫克/毫升： 20-30分钟，在室温下</td></tr></tbody></table> 表2。用于气管酶消化酶类。

isolation of basal cells and submucosal gland duct cells from mouse trachea

大航空公司直接与环境接触，因此容易受到伤害的毒素和传染性我们呼吸的1。大航空公司，因此需要一个有效的修复机制来保护我们的身体。这种修复过程发生在呼吸道的干细胞，这些干细胞气道隔离理解的修复和再生的机制是非常重要的。同样重要的是理解异常修复，可导致气道疾病的2。该方法的目标是从小鼠气管粘膜下腺导管分离出一种新的干细胞群体，将这些细胞在体外和体内模型系统，以确定的修复和再生机制的粘膜下腺体3。生产表明，可用于分离和检测从大的气道3中的管道和基底的干细胞的方法，这将允许我们研究疾病，如囊性纤维化，哮喘和慢性阻塞性肺疾病的气道。目前，有没有隔离的粘膜下腺体导管细胞的方法，也没有在体内模型研究粘膜下腺体的再生。

Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

在这里，我们展示了我们的协议，用于隔离的基础和粘膜下腺体导管细胞从小鼠气管。我们还表明，该方法的干细胞注射到背鼠标脂肪垫，以创建一个<em在体内</em&gt;模式，粘膜下腺体的再生。

小鼠气管基底细胞和粘膜下腺体的导管细胞分离

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

isolation-basal-cells-submucosal-gland-duct-cells-from-mouse

Research

JoVE Journal

Biology

13.3K 次观看.  David Geffen School of Medicine at UCLA. 在这里，我们展示了我们的协议，用于隔离的基础和粘膜下腺体导管细胞从小鼠气管。我们还表明，该方法的干细胞注射到背鼠标脂肪垫，以创建一个

视频: Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

科研

生物学

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...