National Institutes of Health comunhão Fellowship Kirchstein predoctoral AI-64128 (MPM), GM-41514 (MDC), GM-48071 (IP)

1) Remova os dois ossos do fêmur de um rato <ol><li> Use uma tesoura para cortar dissecção muscular e expor o osso fêmur acima e abaixo das articulações (joelho e quadril). Segure o centro do fêmur com uma pinça de dissecção e cortar acima e abaixo das articulações para deixar o máximo de epífise intacta possível. </li><li> Limpar tanto músculo quanto possível, utilizando uma tesoura de dissecção de pequeno porte. Transferência do fêmur em um prato de RPMI. </li><li> Todos os procedimentos devem ser realizados na capa a partir deste ponto, utilizando apenas meios estéreis, instrumentos, ponteiras e placas de cultura. </li></ol> 2) Esterilizar os ossos do fêmur: <ol><li> Na capa, a transferência de ossos da RPMI para um prato de cultura pequeno enchido com etanol a 70% sobre o gelo. </li><li> Permitir que os ossos de molho em álcool por 5 minutos no gelo. </li></ol> 3) Recolha de medula óssea em condições estéreis: <ol><li> Transferência de ossos para um prato de cultura contendo pequenas estéril filtrado meios de cultura primária DC. </li><li> Com uma pinça estéril, segure o osso sobre um prato descartar ou tubo, e com uma tesoura esterilizada, cortar cada epiphesis (extremidades do osso) para fora. O corte deve expor a medula óssea que é vermelho brilhante no centro do osso. </li><li> Com uma seringa estéril (26-28 agulha), sugam cerca de 100-200 mL de meios estéreis. </li><li> Segurar o osso com a pinça estéril sobre um prato de doce de mídia estéril e inserir a agulha em um lado do osso. A posição da ponta da agulha perto do topo do osso e, lentamente, lavar o osso com a mídia estéril. Se você está no centro do osso, a agulha deve deslizar facilmente. </li><li> A medula óssea lava para fora, seja em pequenos pedaços ou como uma peça única. Ele deve ser empurrado para fora do osso e no prato de mídia estéril. </li><li> Repita essa etapa conforme necessário para lavar completamente a medula do osso. </li><li> Quando o osso está limpo, ele será branco e translúcido. </li><li> Repita este procedimento com o osso fêmur outros. Descartar cada osso do fêmur, uma vez que está limpo. </li></ol> 4) Re-suspender e girar as células da medula óssea: <ol><li> Transferência de células para um tubo falcon estéril. </li><li> Se a medula ainda está intacto, muito gentilmente pipeta media cima e para baixo no prato, para quebrar a medula em uma suspensão de células individuais. Isto pode levar alguns minutos e deve ser feito lentamente para evitar matar as células por pura força. </li><li> Células centrífuga como faria com qualquer outras células de mamíferos. </li></ol> 5) Lise Celular: <ol><li> Retire o tubo da centrífuga, após o spin é feito e despeje mídia off (decantar) em um desperdício pequenos (50 mL tubo falcon) na capa. </li><li> Ressuspender suavemente flicking pellet no fundo do tubo. </li><li> Utilizando água filtrada estéril e DPBS 10x ou 10x PBS, realizar uma lise de água para remover as células vermelhas do sangue (hemácias), utilizando os seguintes volumes: <ul><li> 900 mL de água estéril filtrado </li><li> 100 mL DPBS 10x ou 10x PBS </li></ul></li><li> Adicionar 100 mL de segundos 10x PBS 10/05 após a adição da água. É muito importante fazer isso imediatamente, para que apenas as hemácias são lisadas. É uma boa idéia ter as duas pipetas cheio e pronto. </li><li> Adicionar 5 mL - 10 mL de estéril media DC básica. </li></ol> 6) contagem de células: <ol><li> Na capa, o mix mL 6-11 de células da medula óssea rodando suavemente o tubo com uma ligeira inversão, uma inclinação de 45 graus (não para que a mídia toca o topo do tubo) </li><li> Retire 10 mL dos meios de comunicação em um tubo estéril para contar. </li><li> Feche as células e centrifugar novamente. </li></ol> 7) células dendríticas Plating: <ol><li> As células devem ser banhados com uma densidade de 1 x 106/mL. </li><li> Remove as células de centrífuga, a mídia decantar e re-suspensão em células a quantidade adequada de mídia primária DC para revestimento. </li><li> Lugar na cultura de tecidos incubadora. </li></ol> 8) Cuidado Cultura e Maturação: Sempre verifique culturas antes de adicionar mais mídia ou utilizar em qualquer experimentos. Verifique o estado geral de saúde das células e olhar para a contaminação potencial. DIA 0: As células dendríticas banhado. DIA 3: Remove 75% dos meios de comunicação e não adherant células e adicionar mídia primária de volta DC. DIA 6: Depois de Plating célula inicial: Re placa-as células usando o seguinte procedimento: <ol><li> Remova a mídia, que deve conter alguns DCs não adherant neste momento, e colocar em um tubo de 50 mL estéreis falcão, deixando as placas ligeiramente úmido (você não quer que as células aderentes a secar) </li><li> Adicione 3 mM EDTA em PBS para cada prato e deixe descansar por 5 minutos. </li><li> Segure a placa em um ângulo de 45 graus e gentilmente pipeta mídia para cima e para baixo contra a parte inferior da placa gentilmente desalojar não adherant células. </li><li> EDTA / PBS deve tornar-se mais espessa, indicando as células estão sendo coletados a partir do fundo doprato. </li><li> Depois de alguns minutos disso, a mistura de células pode ser transferido para o tubo de 50 mL falcon. </li><li> Girar todas as células, após esse procedimento para cada prato. </li><li> Contagem e placa de as células a uma densidade de 1 x 10 ^ 6 por placa no Media Secundária DC em novas placas de cultura estéril 10 cm. Retorno células para cultura de tecidos incubadora. </li></ol> DIA 11/10 DCs maduras prontas para a estimulação / antígeno de carga. 9) estimulação das células dendríticas: <ol><li> Use LPS (lipopolyscaccharide) em combinação com peptídeo desejado para a activação e carregamento de peptídeo. Dependendo da sua fonte de LPS, condições de estimulação diferentes poderão dar melhores resultados. Além disso, a quantidade desejada de peptídeo para apresentação DC deve ser otimizado. Condições de estimulação neste protocolo foram 100 ng / mL e 100 LPS OVA ug / mL (ovalbumina). </li><li> Antígeno carga / tratar com LPS por 18-24 horas antes da colheita / utilização. </li></ol> Mídia células dendríticas: filtro estéril depois de tudo ter sido adicionado Por favor, consulte a seção de discussão para uma revisão de diferentes condições de cultura de células dendríticas e para o fenótipo resultante célula dendrítica. Estas condições de cultivo são projetados para produzir células dendríticas maduras que rapidamente se casa para os linfonodos de drenagem, 18-24 horas após a injecção subcutânea e antígeno presente de forma eficiente. DC primário Media: filtro estéril depois de tudo ter sido adicionado <ul><li> 500 mL IMDM (remove 55-60 mL de volume final de 500mL) </li><li> 50 mL de calor FBS inativada </li><li> 5 mL de 200 mM L-Gln, concentração final de 2 mM </li><li> 100 UI / ml de penicilina e estreptomicina 100 mg / mL (5 mL de 10.000 Pen IU / mL e 10.000 mg / mL de ações Strep) </li><li> 50 uM B-Me (14,3 M estoque B-Me, diluir 1:100 na capa químicos, use 35 ul por 100 mL de mídia para concentração de 50 micromolar) </li><li> 20-30 ng / mL GM-CSF </li><li> 100-400 UI / mL IL-4 cerca de 10-40 ng / mL </li></ul> * Media SecondaryDC para maturação 100 ng / mL TNF-alfa (add to primário DC media) * Nota: GM-CSF + IL-4 deve produzir células dendríticas imaturas. Usando GM-CSF + IL4 (400 UI / ul) + TNF-alfa (100 ug / mL) irá criar células que se assemelham madura monócitos derivados células dendríticas. Células dendríticas maduras são conhecidos por não levar até o antígeno de forma tão eficiente como células dendríticas imaturas e culturas pode ter endógena TNF-a liberados a partir de células do estroma 4.

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+development+of+murine+dendritic+cells+by+GM-CSF+versus+Flt3+ligand+has+implications+for+inflammation+and+trafficking.">Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.</a> J Immunol. 179, 7577-7584 (2007).</li><li>Inaba, K., Swiggard, W. J., Steinman, R. 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As células dendríticas são mediadores-chave da resposta imune adaptativa ea célula mais eficiente do antígeno apresentando caracterizada até o momento. Métodos para humanos e cultura de células dendríticas do mouse na literatura diferem no tipo de citocinas usadas para influenciar o desenvolvimento de diferentes tipos de células dendríticas. Nomeadamente, a GM-CSF, flt3L, IL4, IL13, TNF-alfa e IFN-gama são utilizadas em diferentes combinações para produzir maduros, imaturos, inflamatórias e steady-state, como medula óssea de...

generation of bone marrow derived murine dendritic cells for use in 2-photon imaging

Vários métodos para a preparação de células dendríticas murinas podem ser encontrados na literatura. Aqui, apresentamos um método que produz mais de 85% CD11c alta células dendríticas em cultura que abriga o linfonodo de drenagem após a injecção subcutânea e antígeno presente nas células T antígeno específico (ver vídeo). Além disso, usamos instrumentos Essen Incucyte para acompanhar a maturação de células dendríticas, onde, no dia 10, a morfologia das células em cultura é típica de uma célula madura dendríticas e% &lt;85 de células são CD11chigh. O estudo da apresentação de antígenos nos linfonodos periféricos por dois fótons de imagem revelou que há três fases distintas de células dendríticas e interação célula T 1, 2. A Fase I consiste de breves contatos serial entre altamente móveis antígeno de células T específicas eo antigénio transportando células dendríticas 1, 2. A fase dois é marcado por contatos prolongados entre antígeno-específica de células T e células dendríticas antígeno tendo 1, 2. Finalmente, a fase III é caracterizada por células T destacando a partir de células dendríticas, recuperando a mobilidade e começam a dividir 1, 2. Este é um exemplo do tipo de antígeno específico interações que podem ser analisados ​​por dois fótons de imagem de antígeno de células-carregados rastreador dye-rotulados células dendríticas.

Watch this Scientific Journal Video about Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging at JoVE.com

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Apresentação de antígenos em órgãos linfóides secundários pelas células dendríticas é crucial para o início da célula T mediada resposta imune adaptativa. Aqui demonstramos a cultura de medula óssea células dendríticas derivadas de camundongos, ativação e de rotulagem para os dois fótons de imagem.

Geração de Medula Óssea Derivado Células Dendríticas murino para uso em dois fótons de imagem

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

generation-bone-marrow-derived-murine-dendritic-cells-for-use-2

Research

JoVE Journal

Biology

31.0K visualizações.  University of California, Irvine (UCI). Apresentação de antígenos em órgãos linfóides secundários pelas células dendríticas é crucial para o início da célula T mediada resposta imune adaptativa. Aqui demonstramos a cultura de medula óssea células dendríticas derivadas de camundongos, ativação e de rotulagem para os dois fótons de imagem.

Vídeo: Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Dissecção e 2-Photon Imagens de linfonodos periféricos em ratos

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Biologia

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Na imagem situ da Thymus Mouse Usando 2-Photon Microscopia

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

Um método para 2-Photon Imagens de Fluxo de Sangue no neocórtex através de uma janela Cranial

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

Vivo em 2-Photon Imagens de cálcio na camada 2 / 3 dos Ratos

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Cultura de células dendríticas mielóides a partir de precursores da medula óssea

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

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

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 were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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 ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing das células hematopoiéticas da medula óssea

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclastos Derivação do mouse Medula Óssea

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

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 ...