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

收获从股骨和胫骨骨髓<ol><li>该过程的第一步是，收获从6股骨和胫骨骨髓 - 12周龄小鼠（我们使用的是BALB / c小鼠，但你可以用此过程中，任何小鼠品系）。首先，安乐死的CO 2吸入（将不会被显示）鼠标。收获从每个后腿股骨和胫骨。然后，用刀片，切删除的髋关节和膝关节股骨的两端，暴露骨髓。 </li><li>以类似的方式，削减的胫骨的两端删除膝，踝关节的邻近地区，并揭露了骨髓。 </li><li>以26号针头与3毫升注射器和填充用RPMI 10％胎牛血清，青霉素，链霉素，和β-巯基乙醇（BME = 100 UM）的补充。使用镊子，缓缴的Petri菜的RPMI媒体骨。骨的一端插入注射器针头，压低柱塞冲洗出骨髓。注射器针头，可以向上和向下移动，骨内，以冲洗掉残留骨髓。 </li><li>重复的过程中，所有的骨头，然后着手建立的文化。 </li></ol>建立文化<ol><li>要建立的骨髓文化，吸刷新骨髓几次分手组织成细胞悬液。 </li><li> 50 mL锥形管上放置一个70微米的过滤器，过滤器，并添加细胞悬液。 </li><li>冲洗培养皿中，用RPMI一次，然后添加到70微米的过滤器冲洗。 </li><li>使用1 mL注射器柱塞橡胶年底，磨70微米的过滤器上的剩余的骨髓片。 </li><li>用RPMI清洗一次过滤器。 </li><li>清洗过滤器后，在5分钟1200转离心细胞悬液。 </li><li> resuspending在PBS清洗细胞沉淀。 </li><li>使用血球计数细胞。我们通常会获得从一只老鼠的骨骼（两股骨加两个胫骨）〜90亿个细胞。细胞计数后，他们再次在5分钟1200转离心。 </li><li>重悬在少量的RPMI和分装到含有足够的RPMI使终浓度为每毫升1万细胞的组织培养瓶中细胞。 </li><li>添加细胞因子，促进发展您感兴趣的细胞类型。在这种情况下，添加细胞因子白细胞介素3（IL - 3 = 10毫微克/毫升）嗜碱性粒细胞和肥大细胞，促进发展。为了获得嗜碱性粒细胞，孵育10天。对于肥大细胞，培育5个星期。当生成肥大细胞，媒体和IL - 3，应每周更换一次。 </li><li>这里是在肥大细胞应如何电镀后出现。这个图像演示肥大骨髓细胞嗜碱性粒细胞和肥大细胞后10天白细胞介素- 3除了的分化。 </li><li>一旦获得所需的细胞类型，进行电一步。 </li></ol>电穿孔细胞<ol><li>要开始的电击一步，决定在培养瓶中的细胞数量。 </li><li>细胞通常在电穿孔密度每毫升10万，转移所需的细胞数量，锥形管，并在5分钟1200转离心管。 </li><li>离心后，吸媒体，用1X PBS清洗细胞，并再次离心5分钟1200转。 </li><li>吸出PBS和添加Bio - Rad公司的基因脉冲电缓冲区，使细胞每毫升10万的细胞悬液。 </li><li> resuspending的细胞后，加入终浓度在10至20微克每毫升所需的质粒DNA。 </li><li>分装150微升到您所选择的井的细胞悬液，在96孔的电板。 </li><li> MXcell板室的电板，并盖上盖子。 </li><li>肥大细胞的转染之前，必须编程电协议进入的MXcell，除非使用预设或存储协议。通过优化实验，我们发现，肥大细胞的转染效率最高发生使用方波脉冲协议。我们将根据板的电条件，提供300V/20ms，350/15ms，350V/20ms和350V/10ms方波脉冲，在2000uF和1000欧姆。一旦协议已经设置并加载到设备上，按“脉冲”electroporate细胞。 </li><li>电完成后，转移到组织培养板的细胞。我们通常将每个150微升电样品以及一个48孔组织培养板与白细胞介素- 3每毫升10毫微克含300微升的RPMI（含10％FCS，青霉素，链霉素，和β-巯基乙醇补充）。细胞孵育过夜在37 ° C，然后在24小时后测定表达和抑制。 </li></ol>陈德良sfection结果成功后，使用质粒GFP的每毫升20微克的电的细胞的荧光显微镜图像显示视频。可使用MXcell电系统的大约30％的转染效率。该系统使您可以改变条件，最大限度地提高您转染效率，同时保持细胞活力。

随着越来越多的基因组信息的出现，并开发新的工具，如siRNA的，使用的有关生理细胞已变得越来越重要，我们进一步了解疾病的途径，蛋白 - 蛋白相互作用和信号转导。主要细胞是直接从组织或体液，并在体外培养。方式，包括通过引入外源遗传物质，这些细胞可以被操纵。这是越来越明显，引入的原代细胞的质粒DNA或siRNA的最有效的方法是电击。 电穿孔公开细胞电脉冲，以瞬时提高细胞膜的通透性，从而使外源核酸进入细胞。这种转染的方法，可以优化，以适应不同类型的细胞/线，因此，可用于转染肥大细胞，以及任何其他骨髓衍生的细胞之间的差异。此外，与病毒介导的转染，电不转染DNA的大小施加限制，也不需要大量的准备。相比之下脂质介导的转染，电可以毒性较低，不会导致转染的核酸内体诱捕。 Bio - Rad公司已经开发出了MXcell电系统，专门为这些细胞。 MXcell允许您以不同条件，最大限度地提高您转染效率和细胞活力。为了优化转染效率和减少细胞死亡，众多的电参数，包括电压，电容，电阻，脉冲长度可以调整和评估。

<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

这是越来越明显，电是最有效的方法，引入的原代细胞的质粒DNA或siRNA。基因脉冲MXcell电系统和基因脉冲电缓冲区是专门开发核酸转染哺乳动物细胞和难以转染的细胞，如小学和干cells.This视频，演示了如何建立从小学小鼠骨髓造血细胞培养，然后准备在MXcell系统的电。我们开始通过分离股骨和胫骨。从股骨和胫骨的骨髓，然后收获和文化都成立。培养的骨髓细胞，然后转染和分析。

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

此过程介绍了如何建立从小鼠骨髓造血的主要细胞培养和使用转基因脉冲MXCell电系统。

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 ...

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Nanyang Technological University

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JoVE Journal

Biology

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视频: 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 对小鼠和人造血祖细胞的高效基因破坏

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

科研

遗传学

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).

隔离和小鼠骨髓的静脉注射单核细胞衍生

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

免疫与感染

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.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

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.

表型分析和分离小鼠造血干细胞和传承的承诺祖

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.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

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.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

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

优化从胸腺和骨髓中分离基质细胞

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.

作者聚焦：使用基质细胞分离进行单细胞测序推进造血研究

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.

从造血器官中分离基质细胞

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.

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 ...

主电穿孔的小鼠骨髓造血细胞培养制备

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此视频中的章节

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

隔离和小鼠骨髓的静脉注射单核细胞衍生

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

表型分析和分离小鼠造血干细胞和传承的承诺祖

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

从造血器官中分离基质细胞

从造血器官中分离基质细胞

从造血器官中分离基质细胞

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