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

La recolección de médula ósea de fémures y tibias <ol><li> El primer paso del procedimiento consiste en la cosecha de médula ósea de los fémures y tibias de un 6 - a la del ratón de 12 semanas de edad (estamos utilizando ratones Balb / c, pero que puede hacer este procedimiento con cualquier cepa de ratón). Para empezar, la eutanasia el ratón por inhalación de CO 2 (no se mostrará). Fémur y la tibia de la cosecha de las patas traseras de cada uno. Luego, con una cuchilla de afeitar, cortar los extremos del fémur para eliminar la cadera y la rodilla y para exponer la médula ósea. </li><li> De manera similar, cortar los extremos de la tibia para eliminar la rodilla y el tobillo y las regiones adyacentes para exponer la médula ósea. </li><li> Tome una jeringa de 3 ml con una aguja de calibre 26 y lo rellenamos con RPMI suplementado con FBS al 10%, penicilina, estreptomicina, y beta-mercaptoetanol (BME = 100 um). Con unas pinzas, sostener el hueso en una placa de Petri que contiene RPMI medios de comunicación. Inserte la aguja de la jeringa en un extremo del hueso y el émbolo para eliminar la médula ósea. La aguja de la jeringa se puede mover arriba y abajo dentro del hueso para eliminar la médula ósea residual. </li><li> Repita el procedimiento con todos los huesos, y luego proceder con el establecimiento de los cultivos. </li></ol> El establecimiento de las Culturas <ol><li> Para establecer los cultivos de médula ósea, una pipeta de la médula ósea lavado de arriba a abajo varias veces para romper el tejido en una suspensión de células. </li><li> Colocar un filtro de 70 micras en la parte superior de un tubo cónico de 50 ml, y añadir la suspensión celular en el filtro. </li><li> Enjuague la placa de Petri una vez con RPMI, a continuación, agregar el enjuague de los 70 filtros de micrón. </li><li> Con el extremo de caucho de un émbolo de una jeringa ml, moler las piezas restantes de médula ósea en la parte superior del filtro de 70 micras. </li><li> Lave el filtro una vez con RPMI. </li><li> Después de haberlo lavado, centrifugar la suspensión celular a 1200 rpm durante 5 minutos. </li><li> Lavar el precipitado celular mediante resuspensión en PBS. </li><li> Contar las células utilizando un hemocitómetro. Por lo general obtener ~ 90 millones de células de los huesos de un ratón (dos fémures más tibias dos). Después de contar las células, que centrifuga de nuevo a 1200 rpm durante 5 minutos. </li><li> Resuspender las células en una pequeña cantidad de RPMI y alícuota en los frascos de cultivo de tejido que contiene suficiente RPMI para que la concentración final es de 1 millón de células por ml. </li><li> Añadir citoquinas para promover el desarrollo de su tipo de célula de interés. En este caso, la citoquina interleuquina-3 (IL-3 = 10 ng / mL) para promover el desarrollo de los basófilos y mastocitos. Para adquirir los basófilos, incubar durante 10 días. De los mastocitos, se incuba durante 5 semanas. Cuando la generación de los mastocitos, los medios de comunicación e IL-3 se debe cambiar una vez por semana. </li><li> Aquí está una mirada a cómo las células del mástil debe aparecer justo después de la siembra. La imagen muestra la diferenciación de las células de la médula mástil en los basófilos y mastocitos 10 días después de la adición de la interleucina-3. </li><li> Una vez que el tipo celular deseado, proceda con el paso de la electroporación. </li></ol> Las células Electroporating <ol><li> Para comenzar la etapa de la electroporación, determinar el número de células en el frasco de cultivo. </li><li> Las células son típicamente electroporated a una densidad de 10 millones por ml, por lo que la transferencia del número de células necesarias para tubos cónicos y centrifugar los tubos a 1200 rpm durante 5 minutos. </li><li> Después de centrifugar, aspirar los medios de comunicación, se lavan las células con PBS 1X, y centrifugar nuevamente a 1200 rpm durante 5 minutos. </li><li> Aspirar el PBS y añadir Bio-Rad Gene tampón de electroporación generador de impulsos para hacer una suspensión celular de 10 millones de células por ml. </li><li> Después de la resuspensión de las células, agregar el ADN del plásmido deseado a una concentración final de 10 a 20 microgramos por mL. </li><li> Alícuota de 150 microlitros de la suspensión de células en los pozos de su elección en una placa de electroporación de 96 pozos. </li><li> Coloque la placa de electroporación en la cámara de placa MXcell y cierre la tapa. </li><li> Antes de la transfección de células cebadas, un protocolo de electroporación debe ser programado en el MXcell a menos que use un protocolo preestablecido o almacenados. A través de experimentos de optimización que hemos descubierto que la mayor eficiencia de la transfección de células cebadas se producen utilizando los protocolos de la onda de pulso cuadrado. Vamos a variar las condiciones de electroporación en la placa para entregar 300V/20ms, 350/15ms, 350V/20ms y 350V/10ms pulsos de onda cuadrada a 2000uF y 1000 ohmios. Una vez que el protocolo se ha establecido y se cargan en el dispositivo, pulse &quot;Pulse&quot; para electroporar las células. </li><li> Después de la electroporación es completa, la transferencia de las células en una placa de cultivo de tejidos. Por lo general la transferencia de cada muestra de 150 microlitros de electroporación de un pozo en una placa de tejido de 48 pocillos con 300 microlitros RPMI (complementado con un 10% de FCS, penicilina, estreptomicina, y beta-mercaptoetanol) a 10 nanogramos por mililitro de interleucina-3. Las células se incubaron durante la noche a 37 ° C, y luego analizaron 24 horas después de la expresión y la represión. </li></ol> TranResultados sfection La imagen de microscopía de fluorescencia de las células después de la electroporación con éxito utilizando los 20 microgramos por mL plásmido GFP se muestra en el video. Utilizando la electroporación MXcell eficiencia de transfección del sistema de alrededor del 30% se puede obtener. El sistema permite variar las condiciones para maximizar la eficiencia de transfección, mientras que el mantenimiento de la viabilidad celular.

A medida que más información sobre el genoma surge y nuevas herramientas como siRNA desarrollados, el uso de células fisiológicamente relevantes se ha convertido cada vez más importante para mejorar nuestra comprensión de los caminos de la enfermedad, las interacciones proteína-proteína, y la transducción de señales. Las células primarias se obtienen directamente de los tejidos o fluidos y cultivadas in vitro. Estas células se pueden manipular de muchas maneras, incluso mediante la introducción de material genético exógeno. Cada vez es más evidente que la forma más eficaz para introducir ADN plásmido o siRNA en las células primarias es la electroporación. Electroporación expone las células a pulsos eléctricos con el fin de aumentar de forma transitoria la permeabilidad de la membrana celular, permitiendo así que los ácidos nucleicos exógenos a entrar en la célula. Este método de transfección se puede optimizar para dar cabida a las diferencias entre los tipos de células diferentes / líneas y, por tanto, puede ser usado para transfectar las células cebadas, así como cualquier otro hueso derivadas de la médula de la célula. Además, a diferencia viral mediada por transfección, electroporación no impone límites en el tamaño de ADN transfectadas, ni requieren una preparación. A diferencia de los lípidos mediada por transfección, electroporación puede ser menos tóxico y no resulta en la captura endosomal del ácido nucleico transfectadas. Bio-Rad ha desarrollado el sistema de electroporación MXcell específicamente para estas células. la MXcell le permite variar las condiciones para maximizar su eficiencia de transfección y viabilidad de las células. Con el fin de optimizar la eficiencia de transfección y reducir al mínimo la muerte celular, una multitud de parámetros de electroporación, incluyendo voltaje, capacidad, resistencia y duración del impulso se puede ajustar y evaluar.

<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

Cada vez es más evidente que la electroporación es la forma más eficaz para introducir ADN plásmido o siRNA en células primarias. El sistema de electroporación Gene Emisores MXcell y Gene tampón de electroporación Emisor se han desarrollado específicamente para transfectar ácidos nucleicos en células de mamíferos y difíciles de transfectar células, como el vídeo cells.This primaria y madre muestra cómo establecer cultivos primarios de células hematopoyéticas de la médula ósea murina , y luego a prepararse para la electroporación en el sistema de MXcell. Comenzamos por el aislamiento del fémur y la tibia. La médula ósea de ambos fémur y la tibia se recolectan y las culturas establecidas. Cultivos de células de médula ósea son entonces transfectadas y analizados.

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 procedimiento se describe cómo establecer cultivos primarios de células hematopoyéticas de la médula ósea murina y es seguida por la transfección utilizando el sistema de electroporación Gene Emisores MXCell.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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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Video: 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.

Interrupción génica altamente eficiente de las células progenitoras hematopoyéticas murinas y humanas 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. ...

Investigación

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).

Aislamiento y la inyección intravenosa de murino médula ósea derivados monocitos

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

Inmunología e Infección

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.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

Biología

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.

El análisis fenotípico y aislamiento de células madre hematopoyéticas murinas y progenitores comprometidos Lineage

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.

Edición del genoma mediado por CRISPR/Cas9 para el estudio de células hematopoyéticas en modelos de enfermedades

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 económico y eficiente para aislar y cultivar células dendríticas derivadas de la médula ósea de ratones

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

Optimización del aislamiento de células estromales del timo y la médula ósea

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 destacado: Avance de la investigación hematopoyética mediante el aislamiento de células estromales para la secuenciación de células individuales

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.

Aislamiento de células estromales de órganos hematopoyéticos

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.

Expresión de proteínas fluorescentes de fusión en murinas médula ósea células dendríticas y 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 ...

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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