美国国立卫生Kirchstein奖学金predoctoral奖学金AI - 64128（MPM）的，GM - 41514（MDC）的GM - 48071（IP）

1）从一个鼠标都股骨骨<ol><li>使用夹层剪刀剪去肌肉和暴露的上方和下方的关节（膝关节和髋关节）的股骨。把握清扫镊子股骨中心和削减的上方和下方的关节离开尽可能完整的骨骺。 </li><li>尽可能使用小夹层剪刀的肌肉清理干净。转移到一盘的RPMI股骨。 </li><li>所有的程序应在油烟机，从这个角度上进行，使用无菌媒体，仪器，移液器和培养皿。 </li></ol> 2）消毒股骨骨头： <ol><li>在油烟机，转移骨骼的RPMI一个小培养皿中充满了70％冰乙醇。 </li><li>允许骨骼中的乙醇浸泡5分钟就冰。 </li></ol> 3）在无菌条件下收集的骨髓： <ol><li>骨骼转移到一个小培养皿中含有无菌过滤的初级直流文化传媒。 </li><li>用无菌镊子，缓缴丢弃的菜或管骨，并用无菌剪刀，切割每个epiphesis（完骨）关闭。切应揭露骨髓，这是鲜艳的红色在骨中心。 </li><li>用无菌注射器（26-28号针头），吸了约100-200毫升的无菌媒体。 </li><li>同在一个无菌媒体的新鲜菜的无菌镊子保持骨和骨的一端插入针。定位针骨上方附近的一角，慢慢地用无菌媒体骨。如果你是在骨的中心，针应该很容易滑动。 </li><li>骨髓清洗，无论是在小件或单件。应该冲出骨和成菜无菌媒体。 </li><li>重复这一步，需要完全洗骨的骨髓。 </li><li>当骨是干净的，这将是白色和半透明的。 </li><li>与其他股骨重复此过程。一旦它是干净的，倒掉每个股骨。 </li></ol> 4）重新挂起和自旋向下骨髓细胞： <ol><li>细胞转移到无菌的猎鹰管。 </li><li>如果骨髓仍保存完好，非常轻柔吸管媒体和菜，分解成单细胞悬液的骨髓。这可能需要几分钟，应该慢慢来，以避免由纯粹的力量杀死细胞。 </li><li>您将任何其他哺乳动物细胞的离心机细胞。 </li></ol> 5）细胞裂解： <ol><li>删除从离心管自旋完成后，倒入一个小废物（50毫升猎鹰管）在引擎盖媒体关闭（倒出）。 </li><li>轻轻弹管底部的悬浮颗粒。 </li><li>使用无菌过滤后的水和10倍DPBS或10X PBS，开展水溶解去除红血细胞（红细胞），使用以下几卷： <ul><li> 900毫升无菌过滤后的水</li><li> 100毫升的10倍DPBS或10倍PBS </li></ul></li><li>加入水后，加入100毫升10倍PBS 5-10秒。这是非常重要的，这样做立即使只有在红细胞裂解。这是一个好主意，有移液器都充满和准备。 </li><li>加入5毫升 - 10毫升无菌直流基本媒体。 </li></ol> 6）计数细胞： <ol><li>在油烟机，6至11毫升的骨髓细胞混合，轻轻摇动管有轻微的反转，45度倾斜（不是让媒体接触抹胸） </li><li>成无菌计数管取出10毫升的媒体。 </li><li>再盖再次细胞和离心机。 </li></ol> 7）电镀树突状细胞： <ol><li>应镀细胞在1 × 106/mL的密度。 </li><li>取出适量的初级直流电镀媒体从离心机的细胞，倒出媒体和重新悬浮细胞。 </li><li>在组织文化的孵化器的地方。 </li></ol> 8）文化关怀和成熟： 请务必检查，然后再添加更多的媒体或在任何实验中使用的文化。检查一般健康的细胞，并寻找潜在的污​​染。 0天：树突状细胞的镀金。 第3天：75％的媒体和非adherant细胞取出，并添加回主直流媒体。 第6天：初始细胞后电镀：重制版的细胞，使用下列程序： <ol><li>取出介质，它应该包含一些非adherant区议会在这一点上，并将其放置在无菌的50毫升猎鹰管，离开板极微湿（你不想贴壁细胞，干出） </li><li> 3 mM EDTA的PBS中添加每块板，并允许坐5分钟。 </li><li>保持在45度角的板轻轻吸管媒体和反对板轻轻驱逐非adherant细胞底部。 </li><li> EDTA / PBS应该成为较厚表明细胞正在收集从底部板。 </li><li>本几分钟后，细胞的混合物，可转移至50毫升猎鹰管。 </li><li>分拆后执行此过程为每个板块的所有细胞。 </li><li>计数板在细胞密度为1 × 10 ^ 6每盘在新的无菌10厘米的培养皿中的直流媒体。返回细胞组织培养孵化器。 </li></ol>天10-11 准备好刺激/抗原负载的成熟DCs。 9）树突状细胞刺激： <ol><li> LPS（lipopolyscaccharide）结合使用所需肽激活和肽加载。根据你的LPS来源，不同的刺激条件下可能会更好的结果。此外，直流介绍肽所需的金额应进行优化。在本议定书的刺激条件分别为100 ng / mL的LPS和100微克/毫升的OVA（卵清蛋白）。 </li><li>负载抗原与收获前18-24小时内毒素/治疗/使用。 </li></ol>树突状细胞介质：无菌过滤后一切已添加请看到不同的树突状细胞的培养条件的审查和讨论导致的树突状细胞的表型部分。这些文化条件成熟树突状细胞，迅速18-24小时后，皮下注射和有效地提呈抗原，淋巴结。 初级直流媒体：无菌过滤后一切已添加 <ul><li> IMDM（删除55-60毫升最终体积为500毫升500毫升） </li><li> 50毫升热灭活胎牛血清</li><li> 5毫升200毫米L -谷氨酰胺，终浓度为2毫米</li><li> 100 IU / ml青霉素和100毫克/毫升链霉素（5毫升10,000 IU / mL的笔和10000毫克/毫升链球菌股票） </li><li> 50 UM乙- ME（14.3 M B - ME股票，稀释化学罩1:100，使用50微摩尔浓度每100毫升媒体35 UL） </li><li> 20-30毫微克/毫升GM - CSF的</li><li> 100-400 IU / mL的IL - 4，大约10 - 40毫微克/毫升</li></ul> SecondaryDC媒体成熟* 100 ng / mL的TNF -α（添加到初级直流媒体） * 注 ：GM - CSF + IL - 4产生未成熟树突状细胞。使用GM - CSF + IL - 4（400 IU / UL）+ TNF -α（100微克/毫升），将创建类似成熟的单核细胞衍生的树突状细胞的细胞。成熟树突状细胞是已知的不占用抗原作为有效的未成熟树突状细胞和文化可能有内源性TNF - a的基质细胞释放4。

<ol>
	<li>Miller, M. J., Safrina, O., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+single+cell+dynamics+of+CD4++T+cell+activation+by+dendritic+cells+in+lymph+nodes.">Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes.</a> J Exp Med. 200, 847-856 (2004).</li><li>Henrickson, S. 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树突状细胞是关键介质的适应性免疫反应和最有效的抗原提呈细胞的特点。文献中对人类和小鼠树突状细胞培养的方法不同类型的细胞因子影响树突状细胞对不同类型的发展。值得注意的是，GM - CSF flt3L，IL4，IL13，TNF -α和IFN -γ是用来在不同的组合，产生成熟的，不成熟的，炎症和稳态像小鼠骨骨髓衍生树突状细胞在体外培养 3-7 。在这里，我们提出了一个简单的方法成熟的小鼠树突状细胞有能力生产归?...

generation of bone marrow derived murine dendritic cells for use in 2-photon imaging

编制小鼠树突状细胞的几种方法可以在文献中发现。在这里，我们提出了一种文化产生大于85％的表面CD11c树突状细胞的方法，回家后皮下注射和目前的抗原，抗原特异性T细胞（见视频）引流淋巴结。此外，我们使用埃森仪器Incucyte跟踪树突状细​​胞的成熟，其中，在第10天，培养细胞的形态是典型的一个成熟的树突状细胞和细胞&lt;85％，是CD11chigh。抗原的外周淋巴结中的演示，通过双光子成像的研究发现，树突状细胞和T细胞相互作用1，2，有三个不同的阶段。第一阶段由高度能动的抗原特异性T细胞和抗原携带树突状细胞的1，2之间的简短的串行接触。第二阶段是标记抗原特异性T细胞和抗原轴承树突状细胞的1，2之间的长期接触。最后，第三阶段的特点是由树突状细胞的分离，恢复活力，并开始划分1，2的T细胞。这是抗原特异性的，可以通过加载抗原细胞跟踪染料标记的树突状细胞的双光子成像分析的交互类型的一个例子。

Watch this Scientific Journal Video about Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging at JoVE.com

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

次级淋巴器官树突状细胞的抗原呈现启动T细胞介导的​​适应性免疫反应的关键。在这里，我们展示了文化的骨髓衍生的小鼠树突状细胞，活化，双光子成像标签。

代骨髓衍生的双光子成像使用的小鼠树突状细胞

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

generation-bone-marrow-derived-murine-dendritic-cells-for-use-2

Research

JoVE Journal

Biology

31.0K 次观看.  University of California, Irvine (UCI). 次级淋巴器官树突状细胞的抗原呈现启动T细胞介导的​​适应性免疫反应的关键。在这里，我们展示了文化的骨髓衍生的小鼠树突状细胞，活化，双光子成像标签。

视频: Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

夹层和小鼠的外周淋巴结的双光子成像

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

科研

生物学

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

在胸腺的鼠标采用双光子显微镜的原位成像

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

通过颅窗口在新皮层的血流量为双光子成像方法

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

归巢的骨髓造血细胞

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...