Este protocolo foi testado e otimizado no laboratório do Dr. David Scadden. Financiamento é fornecido pelo Dana Farber Cancer Institute / Massachusetts General Hospital de Clínicas de Hematologia e Oncologia do Programa Fellowship.

<ol><li> Começamos a experiência homing, extraindo células para serem usadas no experimento. Para esta experiência atual que estamos interessados ​​em descobrir se o homing de medula óssea para os nichos nos ossos longos de animais destinatário é diferente para as células extraídas da medula óssea de animais WT, como C57BL/6J (WT) e aqueles que são transgênicos nocautes para o receptor de Lysophosphatidic 1 (LPAR1) (KO). </li><li> Antes de iniciar o experimento, iremos recolher os materiais que precisamos para este experimento. Em nosso laboratório todas as dissecções do mouse são realizadas em um fluxo laminar de cultura de tecidos, especialmente se as células vão ser transplantados em um animal destinatário, em vez de serem utilizadas para a análise ex-vivo. Começamos colocando uma folha azul dentro do capô. Outros objetos necessários incluem um par de fórceps e uma tesoura afiada que foram autoclavados, um bisturi estéril e descartável cotonetes e álcool. Vamos precisar de PBS modificado e uma placa de 6 estéril bem, um morteiro, azul, 50cc pilão cobertas tubo cônico e um filtro 70uM descartáveis. </li><li> Começamos por tomar um WT e um rato KO. </li><li> Estes ratos são sacrificados por inalação de CO 2 e em seguida mergulhado em 70% isopropranolol por 5 segundos. Este passo impede a contaminação das células de interesse com pele de rato e em nossas mãos não levou a qualquer compromisso no rendimento de células ou resultados experimentais. </li><li> Os ratos são, então, dissecado por sua vez, usando um par de pinças e tesouras afiadas. Nós geralmente começam com o mouse WT como uma questão de rotina. Um snip pequeno é feito na pele ventral do rato que cobre o abdômen e, em seguida, a pele é esticada usando as mãos. A pele sai nos membros posteriores com facilidade. O fêmur ea tíbia são, então, extraído tomando cuidado para não desalojar a cabeça do fêmur como este contém uma grande quantidade de medula óssea. Os membros superiores são destacados de torso do animal pela simples aplicação de tração para eles, mantendo o tronco imóvel. Isso remove os membros superiores como uma peça. Os úmeros são então extraídos os membros superiores. </li><li> Os ossos retirados são colocados em PBS modificado que foi adicionado a 6 pratos bem. PBS modificado é feita pela adição de 2 ml de calor soro fetal bovino inativado e 2 cc de EDTA 0,5 M PH8. Esta solução é então filtrada através de um filtro de 0,45 uM Nalgene e pode ser armazenado a 4 graus Celsius por pelo menos um mês. </li><li> É essencial para rotular os poços da placa, a fim de ser capaz de identificar WT e KO ossos corretamente. </li><li> As carcaças mouse são removidos e eliminados off. </li><li> A nova planilha é colocado no capô e os ossos são limpos usando um bisturi descartáveis. </li><li> Os ossos limpos são colocados um rato em um momento no almofariz. Dois ml de PBS modificado é adicionado à argamassa e os ossos são esmagados utilizando um pilão. É importante primeiro fragmento dos ossos com 10 a frente e para trás dos movimentos da mão e, em seguida, pulverize-as com movimentos rotatórios circular. Eu costumo fazer 50 no sentido horário, então 50 movimentos anti-horário e assim por diante até que o material deixado na argamassa é de cor branca e do PBS modificado adicionado ao material permanece branco ou transparente. </li><li> Depois de cada conjunto de 50 movimentos circulares esmagamento, nós transferimos o líquido da argamassa para a 70 uM filtro colocado no topo de um tubo de 50 ml. Não se esqueça de adicionar 2 ml de PBS para a argamassa depois de cada transferência ao filtro. Você não quer tentar pulverizar ossos secos ou deixe-os secar por qualquer período de tempo que isso levará a apoptose de células. </li><li> Uma vez que o material a argamassa torna-se branco, o material do filtro é transferido para a argamassa eo esmagamento é novamente executada. Isso garante que as células do filtro não são desperdiçados. Normalmente demora um ou dois conjuntos de circular movimentos rotatórios para novamente fazer o material na argamassa na cor branca no momento em que a extração de células está completa. </li><li> A solução no tubo cônico agora é centrifugado (lembre-se de manter as células do WT e mouse KO separado. Daí você terá dois tubos diferentes (uma com células WT, o outro com células KO). Centrifugar a 1200 rpm por 10 minutos. </li><li> Aspirar fora do sobrenadante usando pipetas aspirador separado para cada tubo. Adicione 2 cc de tampão de lise ACK para cada pellet. Este tampão de lise deve ser estéril. Incubar no gelo por 5 minutos. </li><li> Adicionar 8 ml de PBS modificado a cada tubo. Levar 10 ul de cada tubo e pipetar em dois poços separados em uma placa de 96 poços. Colocar os tubos na centrífuga e girá-los a 1200 rpm por 10 minutos. </li><li> Enquanto os tubos estão girando, adicionar 30 ul de tryphan corante azul e 60 ul de PBS modificado a cada poço da placa de 96 poços que transporta as células de interesse. </li><li> Adicionar 10 ul da mistura (por favor, misture bem por pipetagem cima e para baixo) para um hemocitômetro e contar as células nas quatro praças exterior. </li><li> A contagem de células em cada amostra por ml é o número de células contadas, assim, dividido por 4 e multiplied por 100 000. </li><li> Uma vez que a centrifugação for concluída, remova os tubos da centrífuga e ressuspender as células em PBS sem cálcio, magnésio ou glutamina L a uma concentração de 20 milhões de células por ml. Você provavelmente vai ter cerca de 5 ml de volume para o WT e 5 ml de volume para o animal KO. A partir desse passo, vamos estar usando PBS, PBS não modificados para o protocolo. </li><li> A cada tubo adicionar DII, um corante de coloração de células, para alcançar uma concentração final de 5um. Vortex imediatamente por 10 segundos. Incubar a 37 graus por 10 minutos e evitar a exposição à luz. </li><li> Encher os tubos com PBS e girar por 10 minutos a 1200 rpm. </li><li> Após a centrifugação, aspirar o sobrenadante e ressuspender as células em PBS a 40 milhões de células por ml. </li><li> Carga 500 ul em 1 cc seringas com agulha 27G (5 seringas para o mouse WT e 5 seringas para os camundongos KO). Pode ser prudente para preparar 6 seringas para cada coorte. Ter um extra pode ajudar como injeções cauda veia que vamos fazer a seguir pode ser complicado. </li><li> Cubra com papel alumínio as seringas, a fim de manter a DII se desintegre. </li><li> Injetar 500 ul da mistura de células em cada animal destinatário; (5 para cada coorte). </li><li> Estes animais precisam ser irradiados com 9 Gy de irradiação 4-24 horas antes da injeção. </li><li> 48 horas após a injeção, a medula óssea a partir da colheita de membros posteriores de cada animal destinatário. </li><li> Processo como descrito acima para os doadores. Ressuspender as células de cada animal em 1 cc de PBS. Obter uma contagem de células utilizando um hemocitômetro. </li><li> Leia no citômetro de fluxo LSR II da BD ou um instrumento equivalente. O resultado pode ser expressa como a percentagem ou número absoluto de células na medula óssea, que são analisados ​​DII positivo. </li></ol>

<ol>
	<li>Adams, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+engraftment+at+the+endosteal+niche+is+specified+by+the+calcium-sensing+receptor.">Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.</a> Nature. 439 (7076), 599-603 (2006).</li><li>Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+hematopoiesis.">Chemokines in hematopoiesis.</a> Curr Opin Hematol. 15 (1), 49-58 (2008).</li></ol>

Homing é o processo pelo qual células da medula óssea, incluindo as células-tronco, as células progenitoras e células diferenciadas, o seu caminho para a medula óssea depois de ser injetado na corrente sanguínea ou na cavidade da medula óssea de camundongos. Como é óbvio a partir da definição, um cientista pode optar por estudar o homing das células-tronco hematopoiéticas, células progenitoras ou células diferenciadas identificados por imunofenotipagem e isoladas por separação de células. Normalmente...

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<td>DPBS (Dulbecco&#x2019;s Phosphate Buffered Saline</td>
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homing of hematopoietic cells to the bone marrow

Homing é o fenômeno pelo qual transplantadas células hematopoiéticas são capazes de viajar de e para enxertar ou estabelecer residência na medula óssea. Chemomkines vários e receptores estão envolvidos no homing das células-tronco hematopoiéticas. [1, 2] Este documento descreve o protocolo clássico homing utilizados em estudos de células-tronco hematopoiéticas. Em geral, isso envolve isolar a população de células cuja homing precisa ser investigada, manchando esta população com um corante de juros e injetar essas células na corrente sanguínea de um animal destinatário. O animal é então destinatário sacrificados em um tempo pré-determinado após a injeção e da medula óssea avaliados para a percentagem ou número absoluto de células que são positivos para a tintura de interesse. Em um dos esquemas mais comuns experimental, a eficiência homing das células hematopoiéticas de dois animais geneticamente distintos (um animal tipo selvagem e fora knock-correspondente) é comparada. Este artigo descreve o protocolo de células hematopoiéticas homing no quadro de como o experimento.

Watch this Scientific Journal Video about Homing of Hematopoietic Cells to the Bone Marrow at JoVE.com

Homing of Hematopoietic Cells to the Bone Marrow

Este artigo descreve um protocolo usado para estudar o homing das células hematopoiéticas para seus nichos na medula óssea.

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 ...

homing-of-hematopoietic-cells-to-the-bone-marrow

Research

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Biology

29.3K visualizações.  MGH - Massachusetts General Hospital. Este artigo descreve um protocolo usado para estudar o homing das células hematopoiéticas para seus nichos na medula óssea.

Vídeo: Homing of Hematopoietic Cells to the Bone Marrow

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolamento e Análise de Células-Tronco Hematopoéticas da Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Biologia

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 ...

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

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% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

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% ...

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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirus mediada Durante Knockdown Ex Vivo Eritropoese de Células-Tronco Hematopoéticas

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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 ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Isolamento, purificação e diferenciação de precursores de osteoclast da medula óssea de ratos

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

Dinâmica de formação de propulato de plantas de medula óssea fresca

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...