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

A colheita de medula óssea de fêmures e tíbias <ol><li> O primeiro passo do procedimento é a colheita de medula óssea de fêmures e tíbias o de um 6 - a 12 semanas de mouse antigo (que estamos usando camundongos BALB / c, mas você pode fazer este procedimento com qualquer tensão mouse). Para começar, euthanize o mouse por inalação de CO 2 (não irá ser exibido). Fêmur e tíbia colheita das pernas traseiras de cada um. Então, com uma lâmina de barbear, cortar cada extremidade do fémur para remover o quadril e joelho e para expor a medula. </li><li> De forma semelhante, corte cada extremidade da tíbia para remover o joelho eo tornozelo regiões adjacentes e para expor a medula. </li><li> Pegue uma seringa de 3 mL com uma agulha de calibre 26 e preenchê-lo com RPMI suplementado com SFB 10%, penicilina, estreptomicina e beta-mercaptoetanol (BME = 100 uM). Usando uma pinça, segure o osso por uma placa de Petri contendo RPMI mídia. Inserir a agulha da seringa em uma das extremidades do osso e pressionar o êmbolo para expulsar da medula óssea. A agulha da seringa pode ser movida para cima e para baixo dentro do osso para expulsar medula residual. </li><li> Repita o procedimento para todos os ossos, e então proceder com o estabelecimento das culturas. </li></ol> Estabelecer culturas <ol><li> Para estabelecer as culturas de medula óssea, pipetar a medula óssea vermelha para cima e para baixo várias vezes para quebrar o tecido em uma suspensão de células. </li><li> Colocar um filtro de 70 micron em cima de um tubo cônico de 50 mL, e adicione a suspensão de células para o filtro. </li><li> Lavar a placa de Petri uma vez com RPMI, em seguida, adicione a água para o filtro 70 mícron. </li><li> Usando o final de borracha de uma seringa de 1 mL êmbolo, moer os pedaços restantes de medula óssea na parte superior do filtro micron 70. </li><li> Lavar o filtro com um tempo RPMI. </li><li> Depois de lavar o filtro, centrifugar a suspensão celular a 1200 rpm por 5 minutos. </li><li> Lave o pellet celular pela ressuspensão em PBS. </li><li> Contar as células usando um hemocitômetro. Normalmente, obter a 90 milhões de células de ossos um rato (dois fêmures e duas tíbias). Após a contagem das células, centrifugar novamente a 1200 rpm por 5 minutos. </li><li> Ressuspender as células em uma pequena quantidade de RPMI e alíquota-los em frascos de cultura de tecidos contendo RPMI suficiente para que a concentração final é de 1 milhão de células por ml. </li><li> Adicionar citocinas para promover o desenvolvimento do seu tipo de célula de interesse. Neste caso, a citocina interleucina-3 (IL-3 = 10 ng / mL) é adicionado para promover o desenvolvimento de basófilos e mastócitos. Para adquirir basófilos, incubar por 10 dias. Para mastócitos, incubar por 5 semanas. Ao gerar mastócitos, a mídia e IL-3 deve ser trocada uma vez por semana. </li><li> Aqui está uma olhada em como mastócitos deve aparecer apenas após o plaqueamento. Esta imagem demonstra a diferenciação de células da medula mastro em basófilos e mastócitos 10 dias após a adição de interleucina-3. </li><li> Uma vez que o tipo de célula desejada é obtida, prossiga com o passo eletroporação. </li></ol> Células Electroporating <ol><li> Para começar a etapa de eletroporação, determinar o número de células no frasco de cultura. </li><li> As células são tipicamente electroporated a uma densidade de 10 milhões por mL, então transferir o número de células necessárias para tubos cônicos e centrifugar os tubos a 1200 rpm por 5 minutos. </li><li> Após a centrifugação, aspirar a mídia, lavar as células com PBS 1X, e centrifugar novamente a 1200 rpm por 5 minutos. </li><li> Aspirar o PBS e adicionar Bio-Rad Gene tampão de eletroporação Pulser para fazer uma suspensão de células de 10 milhões de células por mL. </li><li> Depois de ressuspender as células, adicionar o DNA desejado plasmídeo em uma concentração final de 10 a 20 microgramas por mL. </li><li> Alíquota de 150 microlitros da suspensão de células em poços de sua escolha em uma placa de eletroporação de 96 poços. </li><li> Coloque a placa na câmara de eletroporação placa MXcell e feche a tampa. </li><li> Antes de transfecção de mastócitos, um protocolo de eletroporação deve ser programado no MXcell menos usando um protocolo preestabelecido ou armazenados. Através de experimentos de otimização descobrimos que a mais alta eficiência de transfecção de mastócitos ocorrem usando protocolos de pulso de onda quadrada. Vamos alterar as condições de eletroporação na placa para entregar 300V/20ms, 350/15ms, 350V/20ms, e pulsos de onda quadrada na 350V/10ms 2000uF e 1000 ohms. Uma vez que o protocolo foi definido e carregado para o dispositivo, pressione &quot;Pulse&quot; para electroporate as células. </li><li> Depois de eletroporação é completo, a transferência das células para uma placa de cultura de tecidos. Normalmente, a transferência de cada amostra de 150 microlitros eletroporação a um poço em uma placa de cultura de 48 poços contendo 300 microlitros tecido RPMI (suplementado com 10% FCS, penicilina, estreptomicina e beta-mercaptoetanol) com 10 nanogramas por ml de interleucina-3. As células são incubadas overnight a 37 ° C, em seguida, analisadas 24 horas depois de expressão e supressão. </li></ol> TranResultados sfection A imagem de microscopia fluorescente das células após a eletroporação bem sucedido usando 20 microgramas por mL GFP plasmídeo é mostrado no vídeo. Usando o eletroporação MXcell a eficácia do sistema de transfecção de cerca de 30% pode ser obtida. O sistema permite variar as condições para maximizar a eficiência de transfecção, mantendo a viabilidade das células.

À medida que mais informação genômica emerge e novas ferramentas, como siRNA são desenvolvidos, o uso de células fisiologicamente relevante se tornar cada vez mais importante para promover nossa compreensão das vias de doença, interações proteína-proteína, e transdução de sinal. As células primárias são obtidas diretamente a partir de tecidos ou fluidos e cultivadas in vitro. Estas células podem ser manipuladas em uma série de maneiras, incluindo através da introdução de material genético exógeno. É cada vez mais evidente que a maneira mais eficaz para introduzir DNA plasmídeo ou siRNA em células primário é eletroporação. Eletroporação expõe as células a pulsos elétricos, a fim de aumentar a permeabilidade transitória da membrana celular, permitindo assim exógenos ácidos nucléicos para entrar na célula. Este método de transfecção pode ser otimizada para acomodar as diferenças entre os diferentes tipos de células / linhas e, portanto, pode ser usado para transfecção mastócitos, bem como de qualquer outro osso de células derivadas da medula. Além disso, ao contrário viral mediada transfecção, eletroporação não impõe limites no tamanho do DNA transfectado, nem requerem uma preparação extensiva. Em contraste com lipídica mediada por transfecção, eletroporação pode ser menos tóxico e não resultar em aprisionamento endossomal do ácido nucléico transfectadas. Bio-Rad foi desenvolvido o sistema de eletroporação MXcell especificamente para estas células. o MXcell permite variar as condições para maximizar sua eficiência de transfecção e viabilidade celular. A fim de otimizar a eficiência de transfecção e minimizar a morte celular, uma multiplicidade de parâmetros eletroporação incluindo tensão, capacitância, resistência e comprimento do pulso pode ser ajustada e avaliados.

<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

É cada vez mais evidente que eletroporação é a forma mais eficaz para introduzir DNA plasmídeo ou siRNA em células primárias. O Gene Pulser sistema de eletroporação MXcell e Gene tampão de eletroporação Pulser foram desenvolvidos especificamente para transfecção ácidos nucleicos em células de mamíferos e de difícil transfecção de células, tais como vídeo primário e cells.This-tronco demonstra como estabelecer culturas primárias de células hematopoiéticas da medula óssea de camundongos , e depois prepará-los para eletroporação no sistema MXcell. Começamos isolando fêmur e tíbia. Medula óssea de ambos os fêmur ea tíbia são, então, colhidas e culturas são estabelecidas. Células da medula óssea cultivadas são, então, transfectadas e analisados.

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

Este procedimento descreve como estabelecer culturas primárias de células hematopoiéticas da medula óssea de camundongos e é seguido por transfecção usando o Gene Pulser sistema de eletroporação MXCell.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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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Vídeo: 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.

Disrupção do Gene altamente eficiente de células progenitoras hematopoiéticas murino e humano por 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. ...

Genética

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).

Isolamento e injecção intravenosa de Murino de Medula Óssea Derivado monócitos

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

Imunologia e Infecção

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.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

Biologia

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.

Análise fenotípica e Isolamento de Células-Tronco Hematopoéticas Murino e Lineage comprometidos Progenitores

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.

Edição do genoma mediado por Lentivirus CRISPR/Cas9 para o estudo de células hematopoiéticas em modelos de doenças

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.

Protocolo Econômico e Eficiente para Isolar e Culturing Células Dendríticas Derivadas da Medula Óssea de camundongos

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

Otimizando o isolamento de células estromais do timo e da medula óssea

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.

Autor em destaque: Avançando na pesquisa hematopoiética usando isolamento de células estromais para sequenciamento de células únicas

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.

Isolamento de células estromais de órgãos hematopoiéticos

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.

Expressão de proteínas da fusão fluorescente em murino derivadas da medula óssea as células dendríticas e macrófagos

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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