このプロトコルは、デビッドスカデンの研究室でテストされ最適化されていました。資金は、ダナファーバー癌研究所/マサチューセッツ総合病院臨床血液腫瘍フェローシッププログラムによって提供されます。

<ol><li>我々は実験で使用するセルを抽出することによりホーミング実験を始める。レシピエント動物の長い骨の隙間に全骨髄のホーミングなどC57BL/6J（WT）及びトランスジェニックいるかを確認することがWTの動物から抽出した骨髄細胞ごとに異なる​​場合は、この現在の実験のために我々が調べることも検討されてリゾホスファチジン受容体1（LPAR1）（KO）のためのノックアウト。 </li><li>我々が実験を開始する前に、我々はこの実験のために必要な資料を収集します。私たちの研究室ではすべてのマウスの解剖は、細胞がレシピエント動物に移植しようとしている代わりに、 生体外分析のために使用されている場合は特に層流の組織培養フード内で行われている。私たちは、フード内ブルーシートを置くことによって始めます。必要に応じて他のオブジェクトには、ピンセットとオートクレーブされているハサミ、滅菌使い捨てメスとアルコール綿棒などがあります。私たちは、修正されたPBSと滅菌6ウェルプレートを必要とする、乳鉢、乳棒、50ccの青は円錐管と70uM使い捨てフィルターを突破した。 </li><li>我々一WT一KOマウスを取ることから始めます。 </li><li>これらのマウスは、CO 2吸入により安楽死させ、その後5秒間isopropranolol 70％に浸漬されています。このステップでは、細胞や実験結果の収率のいずれかの妥協につながっていないマウスの毛皮で、私たちの手で目的の細胞の混入を防ぎます。 </li><li>マウスは、鉗子とはさみのペアを使用して順番に切開されています。我々は通常、ルーチンの問題として、WTマウスで始まります。小さな断片は、腹部を覆うマウスの腹側皮膚で作られ、皮膚は、手を使って拡大されます。皮膚は簡単に後肢をオフにしています。大腿骨と脛骨が、これは骨髄を大量に含んでいるとして大腿骨頭が剥がれないように注意しながら抽出されます。上肢は体幹が動かない維持しながら、単にそれらにトラクションを適用することにより、動物の胴体から切り離されます。これは一体として上肢を削除します。上腕骨は、上肢から抽出されます。 </li><li>削除された骨を6ウェルプレートに追加されている修正されたPBS中に配置されます。変性PBSは、熱不活性化ウシ胎児血清およびEDTA、0.5M PH8の2 ccの2 mlを添加することにより行われます。この溶液をNalgene 0.45μMのフィルターを通して濾過され、少なくとも月に摂氏4度で保存することができます。 </li><li>それは正確にWTとKOの骨を識別できるようにするためにプレートのウェルにラベルを付けることが不可欠です。 </li><li>マウスの死体は削除され、オフに配置されている。 </li><li>新しいシートは、フード内に配置され、骨は使い捨てメスを使用して掃除している。 </li><li>きれいに骨がモルタルで一度にマウスを配置されます。修正PBSの2mlを乳棒を用いてモルタルと骨が粉砕されているために追加されます。それは10でその後にと乳棒のあちこちの動きと骨が円形の回旋の動きとそれらを粉砕最初のフラグメントに重要です。私は通常、50右回り、後、50反の動きを行うので、乳鉢で残った材料は、色および変更されたPBS中で白になるまで、上に白または透明のままの材料に追加。 </li><li> 50円形の粉砕の動きのすべてのセットの後、我々は、モルタルから50 mlコニカルチューブの上に置かれた70μMのフィルターに流体を転送する。フィルタへのすべての転送後にモルタルにPBSを2mlを追加することを忘れないでください。これが細胞のアポトーシスにつながるとして試してみて、粉砕、乾燥骨をまたは任意の期間のためにそれらが乾燥したままにしたくない。 </li><li>モルタルの材料は、白色になると、フィルターの材料は、モルタルに転送され、破砕が再び実行されます。これは、フィルタのセルが浪費されないことを保証します。通常は、再び、細胞の抽出が完了している時に色の白いモルタルの材料を作るために円形の回旋の動きの1つまたは2つのセットを取ります。 </li><li>コニカルチューブ内の溶液は、現在遠心分離し（WTと別のKOマウスから細胞を維持することを忘れないでください。従って、2つの異なるチューブ（WT細胞を持つもの、KO細胞と他の）持っている。10 1200rpmで遠心分。 </li><li>各試験管に別々のアスピレーターのピペットを使用して上清を吸引除去する。各ペレットにACK溶解緩衝液2 Ccを追加。この溶解バッファーは、滅菌する必要があります。氷上で5分間インキュベートする。 </li><li>各チューブに変更されたPBSの8 Ccを追加。 96ウェルプレートに2つの別々のウェルに各チューブとピペットから10 ulを取る。遠心機でチューブをセットし、10分間1200rpmで、それらをスピン。 </li><li>チューブが回転している一方で、目的の細胞を運ぶ96ウェルプレートの各ウェルにtryphan青色色素の30 ULおよび変性PBS 60 ulを追加。 </li><li>血球計算板にこの混合物10μlのを（ピペッティングでよく混ぜてください）​​を追加し、4つの外側の正方形のセルを数える。 </li><li> mlあたり各サンプルの細胞数は4とmultiplieで割ったため、計数した細胞の数です。100 000によるD。 </li><li>遠心分離が完了すると、遠心機からチューブを外し、ミリリットルあたり20万細胞の濃度で、カルシウム、マグネシウムまたはLのグルタミンなしPBSで細胞を懸濁します。あなたは可能性がKO動物のためのWTと容積5mlのための容積の約5mlを持つことになります。我々はPBSを使用するこのステップ以降は、プロトコルのためにPBSを変更されません。 </li><li>各チューブに5uMの最終濃度を達成するためにDII、細胞の染色色素を、追加してください。 10秒直ちにボルテックス。 10分間37℃でインキュベートすると光への暴露を避ける。 </li><li> 1200rpmで10分間PBSとスピンでチューブを埋める。 </li><li>遠心後、上清を吸引し、MLあたり40万セルにPBSで細胞を懸濁します。 </li><li> 27G針（WTマウスとKOマウスは5シリンジ、5シリンジ）で1 ccのシリンジに500μlのをロード。それは、各コホートに6シリンジを準備するのが賢明かもしれません。我々は次に何をしようとしている尾静脈注射はトリッキーなことができるように余分なものを持つことに役立ちます。 </li><li>崩壊からDIIを維持するために、アルミホイルでシリンジをカバーしています。 </li><li>各受信者の動物への細胞の混合物を500 ulを注入し、（各コホートの5）。 </li><li>これらの動物は、4月24日時間注射前に照射の9 Gyで照射する必要があります。 </li><li>注射後48時間、各受信者の動物の後肢から収穫の骨髄。 </li><li>ドナーのために上記のようなプロセス。 PBSの1ccのに各動物からの細胞を再懸濁します。血球計を用いて細胞数を取得します。 </li><li> BDからLSR IIフローサイトメーターまたは同等の楽器をお読みください。結果はパーセンテージまたはDII肯定的に分析骨髄中の細胞の絶対数として表現することができます。 </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>

それによって幹細胞、前駆細胞と分化細胞を含む骨髄細胞、それらの方法は、骨髄へのホーミングには、血流や骨髄のマウスの骨髄腔に注入された後のプロセスです。定義から明らかなように、科学者は、造血幹細胞、前駆細胞または免疫表現型によって識別されると細胞選別によって単離された分化細胞のホーミングを勉強することを選んだことがあります。一般的に科学者は、系統マー?...

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<td>DPBS (Dulbecco&#x2019;s Phosphate Buffered Saline</td>
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<td>Sterile Fetal Bovine Serum</td>
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homing of hematopoietic cells to the bone marrow

ホーミングは、移植された造血細胞へ旅行し、生着または骨髄中の住居を確立することができるする現象です。様々なchemomkinesと受容体は造血幹細胞のホーミングに関与している。 [1、2] 本論文では、造血幹細胞の研究で使用される古典的なホーミングプロトコルの概要を説明します。一般に、これは、そのホーミングニーズ調査される細胞集団を分離する興味の染料でこの集団を染色し、レシピエント動物の血流中にこれらの細胞を注入する。レシピエント動物は、注射や関心の染料のための陽性の細胞のパーセンテージまたは絶対的な数について評価骨髄後所定の時間で屠殺される。最も一般的な実験的手法の一つでは、2つの遺伝的に異なる動物（野生型動物と対応するノックアウト）から造血細胞のホーミング効率が比較されます。この記事では、実験などの枠組みの中で造血細胞ホーミングプロトコルについて説明します。

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

この記事では、骨髄中の彼らのニッチへの造血細胞のホーミングを研究するために使用されるプロトコルについて説明します。

骨髄の造血細胞のホーミング

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 ビュー.  MGH - Massachusetts General Hospital. この記事では、骨髄中の彼らのニッチへの造血細胞のホーミングを研究するために使用されるプロトコルについて説明します。

ビデオ: 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.

プラセンタから造血幹細胞の単離と解析

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. ...

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.

骨髄前駆細胞から骨髄樹状細胞の培養

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.

2光子イメージングで使用するための骨髄由来マウス樹状細胞の世代

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.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

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.

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

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.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

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.

マウス骨髄からの破骨細胞の導出

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.

マウス骨髄由来の樹状細胞とマクロファージの蛍光融合タンパク質の発現

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.

ラット骨髄からの破骨細胞前駆体の分離・精製・分化

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.

マウスの新鮮な骨髄のプロ血小板形成ダイナミクス

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