这个协议进行了测试，并在实验室博士大卫Scadden进行了优化。 Dana Farber癌症研究所/麻省总医院临床血液学和肿瘤学奖学金计划提供资金。

<ol><li>我们开始提取在实验中使用的细胞归巢的实验。对于这个目前的实验中，我们感兴趣的是找出如果收件人动物长骨龛的全骨髓寻的是不同的从野生型动物，如C57BL/6J（WT）中提取的骨髓细胞和那些转基因为溶血磷脂受体1（LPAR1）（KO）击倒。 </li><li>在我们开始实验之前，我们将收集的材料，我们这个实验的需要。所有的鼠标在我们的实验室进行解剖是在层流组织培养罩，尤其是当细胞将被移植到收件人的动物，而不是被用于体外分析。我们开始放置一个蓝色的纸罩内。所需的其他对象包括一个钳和一双一双已灭菌的剪刀锋利，一次性无菌手术刀和酒精棉签。我们将需要修改后的PBS和无菌6孔板，一枚迫击炮弹，杵，50CC蓝色荣登锥形管和一个70uM的一次性过滤器。 </li><li>我们开始考虑一个WT和一个正鼠标。 </li><li>这些老鼠是安乐死CO 2吸入，然后蘸在5秒isopropranolol 70％。此步骤可防止用鼠标在我们手中的毛皮和利益细胞的污染并没有导致任何妥协细胞或实验结果的收益。 </li><li>然后解剖老鼠是在使用一对钳和锋利的剪刀之交我们通常开始与野生型小鼠作为例行公事。腹侧皮肤鼠标覆的腹部皮肤，然后使用手伸在一个小剪断。关闭后肢的皮肤很容易产生。股骨和胫骨，然后照顾不打跑股骨的头部，因为这包含了大量的骨髓中提取的。简单地应用牵引，同时保持躯干不动，上肢脱离动物的躯干。这将删除一块上肢。然后提取上肢的肱骨。 </li><li>删除骨骼被放置在改良PBS已加入到6孔板。 PBS是修改加入2毫升热灭活胎牛血清和2毫升EDTA的0.5 mol PH8。该解决方案，然后通过一个NALGENE 0.45 UM过滤器和过滤，可在4摄氏度保存至少一个月。 </li><li>它是必不可少的标签板的井，以便能够正确识别WT和KO骨骼。 </li><li>鼠标壳拆除和处置。 </li><li>一个新的工作表被放置在引擎盖和骨头都​​使用一次性手术刀清理。 </li><li>洗干净的骨头都放在一个鼠标，在砂浆的时候。改性PBS毫升添加到砂浆使用杵和骨头被压碎。 10至杵来回运动，然后骨头粉碎圆形旋转运动，它是重要的第一个片段。我平时50顺时针，然后逆时针旋转50运动，等等，直到留在砂浆材料是白色的颜色和修改后的PBS的材料仍然是白色或透明。 </li><li>每50圆形的破碎动​​作后，我们将流体从砂浆一个50毫升的锥形管上放置一个70微米过滤器。不要忘记添加转移到过滤器后，每2毫升的PBS砂浆。你不想尝试和粉碎干燥的骨骼或离开他们干任何时间的长短，因为这会导致细胞凋亡。 </li><li>在砂浆材料一旦变为白色，过滤材料转移到迫击炮和破碎再次执行。这将确保在过滤器中的细胞不被浪费。通常需要一个或两个圆形旋转运动再次使材料在砂浆的颜色白色，而此时的细胞提取完成。 </li><li>在锥形管的解决方案是离心（请记住保持WT和KO鼠标单独的细胞。因此，你将有两个不同的管（与WT细胞，正细胞）。离心机1200转10分钟。 </li><li>每个管单独使用吸引器吸液管吸上清。 ACK裂解液中加入2毫升，每个颗粒。此裂解液应无菌。在冰上孵育5分钟。 </li><li>每管加入8毫升改性PBS。考虑到在96孔板中的两个单独的井从每管10 UL和吸管。放置在离心管和旋转10分钟1200转。 </li><li>虽然管旋转，添加30 tryphan蓝色染料改性PBS和60 UL UL以及每96孔板中进行细胞。 </li><li> 10 UL这种混合物（请​​混合移液器向上和向下），血球计数细胞外的四个广场。 </li><li>在每个样本每毫升的细胞计数，从而计算细胞数目除以4和multiplieD的100 000。 </li><li>离心完成后，取出离心管和无钙，镁或L谷氨酰胺的浓度每毫升20万细胞的PBS重悬细胞。您将有可能为高山动物的WT和5毫升的体积约5毫升的体积。从这一步开始，我们将用PBS，不修改该协议的PBS。 </li><li>每管添加DII，细胞染色的染料，达到5um的最终浓度。涡立即为10秒。孵育10分钟，在37度，并避免暴露在光线下。 </li><li>填充管，用PBS和自旋为10分钟1200转。 </li><li>离心后，吸上清，在PBS重悬在每毫升40万细胞的细胞。 </li><li> 1毫升注射器装入一个27G的针头（WT鼠标和5 KO小鼠注射器注射器）500 UL。这可能是审慎的准备为每个​​队列6注射器。有一个额外的帮助，为我们下一步准备做的尾静脉注射可能会非常棘手。 </li><li>用铝箔覆盖的注射器，以保持从瓦解的直接投资收益。 </li><li>注入到每个收件人的动物细胞混合UL 500;（5）每个队列。 </li><li>这些动物需要4-24小时注射前9戈瑞的辐射照射。 </li><li>注射后48小时，收获从每个收件人动物的后肢骨髓。 </li><li>过程描述了上面的捐助者。从每个动物的细胞在1毫升的PBS重悬。获取一个细胞计数使用hemacytometer。 </li><li>阅读屋宇署的LSR II流式细胞仪或等效的仪器。可以表示为在分析骨髓细胞的百分比或绝对数量DII积极结果。 </li></ol>

<ol>
	<li>Adams, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+engraftment+at+the+endosteal+niche+is+specified+by+the+calcium-sensing+receptor.">Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.</a> Nature. 439 (7076), 599-603 (2006).</li><li>Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+hematopoiesis.">Chemokines in hematopoiesis.</a> Curr Opin Hematol. 15 (1), 49-58 (2008).</li></ol>

寻的是过程中，骨髓细胞，包括干细胞，祖细胞分化的细胞，他们被注射进入血液流或小鼠骨髓腔后，进入骨髓。由于从定义是明显的，科学家可能选择研究造血干细胞，祖细胞分化的细胞或确定的免疫表型和细胞分选分离的归巢。通常科学家贫宗族标记注入骨髓细胞和祖细胞和干细胞丰富。 利益注入细胞的剂量是在这个实验中的变量。细胞的数量可以改变每个收件人鼠标?...

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homing of hematopoietic cells to the bone marrow

寻的是现象，使移植的造血干细胞是能够前往和嫁接，或建立在骨髓居住。各种chemomkines和受体参与造血干细胞归巢。 [1，2] 本文概述了经典的寻的协议，用于造血干细胞的研究。在一般情况下，这种分离的细胞群的寻的需求进行调查，人口与利益的染料染色和收件人的动物的血液流注入这些细胞。收件人的动物，然后牺牲在一个预先确定的时间，注射后和骨髓的细胞，这些细胞染料的利益积极的百分比或绝对数量评估。在最常见的实验计划，从两个不同的动物基因造血干细胞（野生型动物和相应的基因敲除）的归巢效率是相比。本文介绍了归巢的造血干细胞作为实验的框架协议。

Watch this Scientific Journal Video about Homing of Hematopoietic Cells to the Bone Marrow at JoVE.com

Homing of Hematopoietic Cells to the Bone Marrow

本文介绍了用于研究对他们龛的造血干细胞归巢于骨髓的协议。

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 ...

homing-of-hematopoietic-cells-to-the-bone-marrow

Research

JoVE Journal

Biology

29.3K 次观看.  MGH - Massachusetts General Hospital. 本文介绍了用于研究对他们龛的造血干细胞归巢于骨髓的协议。

视频: Homing of Hematopoietic Cells to the Bone Marrow

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

科研

生物学

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 ...

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

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% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

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% ...

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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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 ...

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 ...

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

小鼠新鲜骨髓外植体的丙酸盐形成动力学

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...