Name: the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation
Uploaded: 2009-01-06T23:59:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation at JoVE.com

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。

<table><tbody><tr><td>Gene Pulser MXcell Electroporation System</td><td>Bio-Rad</td><td>165-2670</td><td></td></tr><tr><td>Gene Pulser Electroporation Buffer</td><td>Bio-Rad</td><td>165-2677</td><td></td></tr></tbody></table>

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 ...

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25.7K ビュー.  Bio-Rad Laboratories, Inc.  ChineseVersion 

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

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 によるマウスおよび人間の造血前駆細胞の高効率遺伝子破壊

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

遺伝学

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

単離とマウス骨髄由来の単球の静脈注射

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

免疫・感染学

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

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 (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

生物学

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

疾患モデルにおける血化細胞の研究のためのレンチウイルスCRISPR/Cas9媒介ゲノム編集

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

マウスから骨髄由来樹状細胞を単離および培養するための経済的かつ効率的なプロトコル

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

胸腺および骨髄からの間質細胞単離の最適化

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

著者スポットライト:シングルセルシーケンシングのための間質細胞単離を用いた造血研究の推進

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

造血器官からの間質細胞の単離

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

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 ...

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

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CRISPR/Cas9 によるマウスおよび人間の造血前駆細胞の高効率遺伝子破壊

単離とマウス骨髄由来の単球の静脈注射

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

疾患モデルにおける血化細胞の研究のためのレンチウイルスCRISPR/Cas9媒介ゲノム編集

マウスから骨髄由来樹状細胞を単離および培養するための経済的かつ効率的なプロトコル

造血器官からの間質細胞の単離

造血器官からの間質細胞の単離

造血器官からの間質細胞の単離

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