大腿骨と脛骨から骨髄を採取<ol><li> 12週齢のマウス（我々はBALB / cマウスを使用しているが、任意のマウス系統では、この手順を行うことができます）に - 手順の最初のステップは、6の大腿骨と脛骨から骨髄を採取することです。開始するには、CO 2吸入（表示されません）でマウスを安楽死させる。それぞれの後ろ足から収穫の大腿骨と脛骨。その後、カミソリの刃で、股関節と膝関節を削除するには、大腿骨の両端をカットして骨髄を公開する。 </li><li>同様の方法では、膝と足首の隣接領域を削除すると骨髄を公開するために脛骨のそれぞれの端をカット。 </li><li> 26ゲージの針3 mLシリンジを取り、10％FBS、ペニシリン、ストレプトマイシン、およびβ-メル​​カプトエタノール（BME = 100 UM）を添加したRPMIで塗りつぶす。ピンセットを使用して、RPMI培地を含むペトリ皿上の骨を保持する。骨の一端に注射針を挿入し、骨髄をフラッシュするためにプランジャーを押し下げる。注射針が残留骨髄を吐き出すのに骨の内部で上下に移動することができます。 </li><li>文化の確立を続行してすべてのボーンのための手順を繰り返し、と。 </li></ol>文化の確立<ol><li>骨髄培養を確立するために、細胞懸濁液中に組織を分割するには、上下に数回フラッシュされた骨髄をピペット。 </li><li> 50 mLコニカルチューブの上に70ミクロンのフィルターを配置し、フィルターに細胞懸濁液を加える。 </li><li> RPMIでペトリ皿を一度洗い流した後、70ミクロンのフィルターにすすぎ追加。 </li><li> 1 mLのシリンジプランジャのゴムのエンドを使用して、70ミクロンのフィルターの上に残っている骨髄の部分を挽く。 </li><li> RPMIでフィルタを1回洗浄する。 </li><li>フィルターを洗浄した後、5分間1200rpmで細胞懸濁液を遠心してください。 </li><li> PBSに再懸濁することにより細胞ペレットを洗浄します。 </li><li>血球計算板を用いて細胞を数える。我々は通常、1つのマウスの骨（二大腿骨と2つの脛骨）から〜90万個の細胞を得る。細胞をカウントした後、5分間1200rpmで再度遠心する。 </li><li> RPMIと注し、その最終的な濃度がmLの当たり1万個の細胞であることを十分にRPMIを含む組織培養フラスコに少量の細胞を再懸濁します。 </li><li>興味のある細胞のタイプの開発を促進するサイトカインを追加。このケースでは、サイトカインのインターロイキン-3（IL - 3 =は10 ng / mL）を好塩​​基球およびマスト細胞の発達を促進するために追加されます。好塩基球を取得するには、10日間インキュベートする。肥満細胞の場合は、5週間インキュベートする。肥満細胞を生成するときに、メディアおよびIL - 3は、週に1回交換する必要があります。 </li><li>ここだけのメッキの後に表示される方法肥満細胞を見ている。この画像は、10日間のインターロイキン- 3を添加した後、好塩基球とマスト細胞にマスト骨髄細胞の分化を示しています。 </li><li>所望の細胞タイプを取得すると、エレクトロポレーションのステップに進みます。 </li></ol> Electroporatingセル<ol><li>エレクトロポレーションのステップを開始するには、培養フラスコ中の細胞の数を決定します。 </li><li>細胞は通常はmLの1千万人もの密度でエレクトロポレーションされているので、必要な細胞数は、コニカルチューブに移して5分間1200rpmでチューブを遠心する。 </li><li>遠心分離した後、1 × PBSで細胞を洗浄し、5分間1200rpmで再び遠心、培地を吸引除去する。 </li><li> PBSを吸引除去し、mL当たり107細胞の細胞懸濁液を作るためにバイオラッドジーンパルサーのエレクトロポレーションバッファーを追加します。 </li><li>細胞を再懸濁させた後、液あたり10〜20マイクログラムの最終濃度で所望のプラスミドDNAを加える。 </li><li> 96ウェルエレクトロプレート上の任意のウェルに細胞懸濁液のアリコートを150マイクロリットル。 </li><li> MXcellプレートチャンバーのエレクトロポレーションプレートを入れ、蓋を閉じます。 </li><li>肥満細胞のトランスフェクションに先立ち、エレクトロポレーションプロトコルは、プリセットまたは保存されているプロトコルを使用していない限り、MXcellにプログラムする必要があります。最適化実験を通して、私たちは、肥満細胞の最も高いトランスフェクション効率は、方形波パルスのプロトコルを使用して発生することを発見した。我々は、2000uFと1000オームで300V/20ms、350/15ms、350V/20ms、および350V/10ms方形波パルスを提供するプレート上にエレクトロポレーション条件を変化させます。プロトコルが設定されており、デバイスにロードされた後、細胞をエレクトロに&quot;パルス&quot;を押してください。 </li><li>エレクトロポレーションが完了したら、組織培養プレートに細胞を移す。我々は通常、mLのインターロイキン- 3当たり10ナノグラム、300マイクロリットルRPMI（10％FCS、ペニシリン、ストレプトマイシン、およびβ-メル​​カプトエタノールを補充した）を含む48ウェル組織培養プレートにウェルにそれぞれ150マイクロリットルのエレクトロポレーションのサンプルを移す。細胞は37℃で一晩インキュベートする° Cは、その後の発現と抑制のために24時間後にアッセイした。 </li></ol>トランsfection結果プラスミドmLのGFPあたり20マイクログラムを使用して成功したエレクトロポレーション後の細胞の蛍光顕微鏡像をビデオに示されています。約30％のMXcellエレクトロ系のトランスフェクション効率を用いて得ることができる。細胞の生存率を維持しながら、システムは、あなたのトランスフェクション効率を最大化するための条件を変化させることができます。

より多くのゲノム情報が出てくるとこのようなsiRNAなどの新しいツールが開発されるように、生理学的に関連する細胞の使用は、病気の経路、タンパク質 - タンパク質相互作用、及びシグナル伝達の理解を促進するためにこれまで以上に重要になっています。初代細胞は、組織又は体液から直接取得し、in vitroで栽培されている。これらの細胞は、外因性遺伝物質の導入などを通じて、さまざまな方法で操作することができます。それは一次細胞にプラスミドDNAやsiRNAを導入する最も効果的な方法は、エレクトロポレーションであることがますます明らかになってきている。 エレクトロポレーションは、一時的にそれによって外因性核酸は、細胞を入力できるように、細胞膜の透過性を向上させるために電気パルスにセルを公開します。トランスフェクションのこのメソッドは、異なる細胞タイプ/ラインと、このように、マスト細胞をトランスフェクトするために使用できるだけでなく、他の骨髄由来細胞の違いに対応するために最適化することができます。さらに、ウイルスの媒介トランスフェクションとは異なり、エレクトロポレーションはトランスフェクトしたDNAのサイズに制限があります、また大規模な準備を必要としません。脂質媒介トランスフェクションとは対照的に、エレクトロポレーションは、毒性の低いことができ、トランスフェクトされた核酸のエンドソーム捕捉されることはありません。 バイオラッドは、これらの細胞のために特別にMXcellのエレクトロポレーションシステムを開発しました。 MXcellは、あなたのトランスフェクション効率と細胞生存率を最大化するための条件を変化させることができます。トランスフェクション効率を最適化し、細胞死を最小限にするためには、電圧、キャパシタンス、抵抗、およびパルス長を含むエレクトロポレーションパラメータの多数を調整し、評価することができます。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gene Pulser MXcell Electroporation System</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2670</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser Electroporation Buffer</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2677</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation

それは、エレクトロポレーションは、プライマリ細胞にプラスミドDNAやsiRNAを導入するための最も効果的な方法であることがますます明らかになってきている。ジーンパルサーMXcellのエレクトロポレーションシステムとジーンパルサーのエレクトロポレーションバッファー特に哺乳動物細胞や、プライマリおよび幹cells.Thisビデオなどの困難なトランスフェクション細胞に核酸をトランスフェクトするために開発されたマウス骨髄からプライマリ造血細胞培養を確立する方法を示していますし、MXcellシステムにおけるエレクトロポレーションのためにそれらを準備する。我々は、大腿骨と脛骨を分離することから始めます。大腿骨と脛骨の両方から骨髄を回収され、文化が確立されています。培養骨髄細胞は、トランスフェクションと分析される。

Watch this Scientific Journal Video about The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation at JoVE.com

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

この手順では、マウス骨髄からプライマリ造血細胞培養を確立し、ジーンパルサーMXCellのエレクトロポレーションシステムを用いてトランスフェクションが続いている方法について説明します。

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 ...

<a class="grey_button" href="/video/1047">
ChineseVersion
</a>

the-preparation-primary-hematopoietic-cell-cultures-from-murine-bone

Research

JoVE Journal

Biology

25.6K ビュー.  Bio-Rad Laboratories, Inc.  ChineseVersion 

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

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

生物学

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

後期ステージショウジョウバエ蛹の脳からの初代神経培養

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

抗酸菌のエレクトロポレーション

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

げっ歯類の新生児から一次分離した中脳ドーパミン細胞培養

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

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.

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

から初代細胞培養ショウジョウバエ原腸胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

基礎脊椎動物の系統からの一次筋原前駆細胞/筋芽細胞培養液の調製

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

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 ...