Bu protokol Dr David Scadden laboratuvar test ve optimize edilmiştir. Finansman, Dana-Farber Kanser Enstitüsü / Massachusetts Genel Hastanesi Klinik Hematoloji ve Onkoloji Burs Programı tarafından sağlanmaktadır.

<ol><li> Biz, deneyde kullanılmak üzere hücrelere açılan homing deney başlar. Alıcı hayvanların uzun kemiklerde nişler tüm kemik iliği homing WT C57BL/6J (WT) gibi hayvanlardan elde edilen kemik iliği hücreleri için farklı ve transgenik olanlar eğer bu deneme için biz bulmaya ilgilenen Lysophosphatidic Reseptör 1 (LPAR1) (KO) nakavt. </li><li> Deney başlamadan önce, bu deney için gerekli malzemeleri toplamak. Hücrelerin yerine ex-vivo analizi için kullanılan bir alıcı hayvan nakledilen olacak laboratuvar, özellikle tüm fare diseksiyonları bir laminer akış doku kültürü kaputu yürütülmektedir. Biz kaput içinde mavi bir levha koyarak başlar. Ihtiyaç duyulan diğer nesneleri forseps bir çift ve otoklavda sterilize edilmiş keskin bir makas, neşter ve alkol steril tek kullanımlık bezlerden içerir. Biz değiştirilmiş PBS ve steril bir 6 plaka, harç gerekecektir, havaneli şart, 50cc mavi konik tüp ve 70uM bir tek filtre tepesinde. </li><li> Biz bir WT ve bir KO fare alarak başlar. </li><li> Bu fareler, CO 2 Solunduğunda ötenazi ve daha sonra 5 saniye boyunca% 70 isopropranolol batırılmış. Bu adım, kirlenmesini fare kürk ve bizim ellerimizde ilgi hücreleri, hücreler ya da deneysel sonuçlar verim herhangi bir uzlaşma değil önler. </li><li> Fareler daha sonra bir çift forseps ve keskin bir makas kullanarak sırayla disseke edilir. Biz genellikle rutin bir mesele olarak WT fare ile başlar. Küçük bir snip, karın ve deri daha sonra ellerini kullanarak gerilir üzerinde fare ventral deri yapılır. Cilt kolayca arka bacaklarda çıkıyor. Femur ve tibia sonra femur başı, bu büyük bir miktar kemik iliği içerdiğinden çıkarmak için özen ayıklanır. Üst ekstremitelerde, gövde hareketsiz tutarak sadece onlara çekiş uygulanarak hayvan gövde ayrıdır. Bu, tek parça olarak üst ekstremitelerde kaldırır. Humeri sonra üst ekstremitelerde ayıklanır. </li><li> Kaldırılan kemikler 6 kuyucuğu eklenmiş olan modifiye PBS yerleştirilir. Modifiye PBS ısı inaktive fetal Bovine Serum ve EDTA 0,5 M pH8 2 cc 2 ml ekleyerek yapılır. Bu çözüm daha sonra bir Nalgene 0.45 um filtre süzülür 4 santigrat derece sıcaklıkta ve en az bir ay süreyle saklanabilir. </li><li> WT ve KO kemikleri doğru tespit edebilmek için plakasını kuyuların etiketlemek için esastır. </li><li> Fare karkaslar kaldırılır ve bertaraf. </li><li> Yeni bir sayfa kaput yerleştirilir ve kemikler tek bir neşter kullanılarak temizlenir. </li><li> Temizlenmeli kemiklerin harcı bir süre tek bir fare yerleştirilir. İki ml modifiye PBS harç eklenir ve kemiklerin bir havaneli kullanarak ezilmiş. Sonra 10 ve havaneli fro hareketleri ve kemikleri dairesel dönme hareketleri ile onları ezebileceği ilk parça için önemli. Ben genellikle 50 saat yönünde, daha sonra 50 saat yönünün tersine hareketleri yapmak ve böylece havanda sol malzeme, renk ve modifiye PBS beyaz kadar beyaz veya şeffaf kalır malzeme eklendi. </li><li> 50 dairesel kırma hareketleri her ayarladıktan sonra, harç, 50 ml konik tüpün üstüne yerleştirilen 70 UM filtre sıvı transferi. Filtre Her aktarımdan sonra harç 2 ml PBS eklemek için ihmal etmeyin. Denemek ve bu hücrelerin apoptozis yol açacak gibi ezebileceği kuru kemiklerin veya uzun bir süre için onlara kuru ayrılmak istemiyorum. </li><li> Harç malzemesi beyaz olduktan sonra, filtre malzemesi harç transfer ve kırma tekrar yapılır. Bu filtre hücreleri israf edilmemesini sağlar. Genellikle hücre çıkarma, hangi zamanda harç beyaz renkli malzeme tekrar yapmak için bir veya iki adet dairesel dönme hareketleri alır. </li><li> Konik tüp çözüm (lütfen WT ve KO fare ayrı hücrelere tutmak için unutmayın. Dolayısıyla 1200 rpm&#39;de 10 için iki farklı boru (KO hücreleri ile WT hücreleri bir diğer). Santrifüj olacak santrifüj dakika. </li><li> Her tüp için ayrı aspiratör pipetler kullanarak süpernatantı aspire. ACK lizis tamponu 2 cc her pelet ekleyin. Bu lizis tamponu steril olmalıdır. 5 dakika boyunca buz üzerinde inkübe edin. </li><li> Her tüpe 8 cc modifiye PBS ekleyin. 96 plaka iki ayrı kuyu içine her tüpten 10 ul ve pipet alın. Santrifüj tüpleri yerleştirin ve 10 dakika 1200 rpm&#39;de onları döndürün. </li><li> Tüpler iplik olmasına rağmen, ilgi hücreleri taşıyan 96 plaka her kuyuya tryphan mavisi 30 ul ve modifiye PBS 60 ul ekleyin. </li><li> Bir hemasitometre ekleyin ve bu karışımı 10 ul (yukarı ve aşağı pipetleme iyice karıştırın lütfen) dört dış kare hücreleri saymak. </li><li> Ml başına her örnek hücre sayımı, 4 ve multiplie bölünmüş böylece sayılır hücrelerinin sayısı100 000 d. </li><li> Santrifüj tamamlandıktan sonra, santrifüj tüpleri kaldırmak ve ml&#39;de 20 milyon hücre bir konsantrasyonda kalsiyum, magnezyum veya L glutamin olmadan PBS içinde hücreleri tekrar süspansiyon haline getirin. Büyük olasılıkla, KO hayvan için WT ve 5 ml hacim hacmi yaklaşık 5 ml olacak. Biz bu adımı itibaren protokol PBS değişmiyor PBS kullanarak olacak. </li><li> Her tüp eklemek DII, bir hücre boyama boya, 5uM son bir konsantrasyon elde etmek için. 10 saniye vorteksleyin. 37 derece 10 dakika inkübe edin ve ışığa maruz kalmasını önlemek. </li><li> Tüpler 1200 rpm&#39;de 10 dakika süreyle PBS ve spin ile doldurun. </li><li> Santrifüj sonra, süpernatant aspirat ve ml&#39;de 40 milyon hücre PBS içinde hücreleri tekrar süspansiyon haline getirin. </li><li> 27G iğne (WT fare ve KO fareler için 5 şırınga için 5 şırınga) ile 1 cc şırınga içine 500 ul yükleyin. Her kohort için 6 şırınga hazırlamak için ihtiyatlı olabilir. Önümüzdeki yapacağız kuyruk ven enjeksiyonları yanıltıcı olabilir gibi ekstra bir tane yardımcı olabilir. </li><li> Dağılmakta DII tutmak için şırınga folyo ile örtün. </li><li> 500 ul her bir alıcı hayvan hücresi karışımı enjekte edilir (her kohort için 5). </li><li> Bu hayvanlar, enjeksiyon için 4-24 saat önce 9 Gy ışınlama ile ışınlanmış olması gerekir. </li><li> Enjeksiyon sonrası 48 saat, her alıcı hayvanın arka bacaklarda hasat kemik iliği. </li><li> Bağışçılar için yukarıda açıklandığı gibi işleyin. PBS 1 cc her hayvan hücreleri tekrar Hemasitometre kullanarak bir hücre sayımı alın. </li><li> BD gelen LSR II akış sitometresi veya eşdeğer bir enstrüman okuyun. Sonuç DII pozitif analiz kemik iliği hücrelerinin yüzdesi ya da mutlak sayısı olarak ifade edilebilir. </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 süreci bu sayede kemik iliği hücreleri kök hücreleri, progenitör hücreler ve farklı hücreler, kan akışı veya farelerde kemik iliği boşluğuna enjekte edildikten sonra kemik iliği onların yol içine dahil. Tanımı açık olarak, bir bilim adamı, hematopoietik kök hücre, progenitör hücreler ya da hücre sıralayarak izole işaretleyicidir tarafından belirlenen ve farklılaşmış hücrelerin homing çalışma seçebilirler. Tipik olarak, bilim adamları, soy belirteçler için tükenmiş kemi...

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<td>DPBS (Dulbecco&#x2019;s Phosphate Buffered Saline</td>
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<td>Sterile Fetal Bovine Serum</td>
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<td>Valley Biomedical, Inc</td>
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<td>0.5M EDTA (pH 8.0)</td>
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<td>Boston BioProducts</td>
<td>BM-150</td>
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<td>Mediatech, Inc</td>
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<td>Vybrant DiD cell-labelling solution</td>
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homing of hematopoietic cells to the bone marrow

Homing nakledilen hematopoetik hücreler ve aşılamak ya da kemik iliği ikamet kurmak mümkün sağlayan bir olgudur. Çeşitli chemomkines ve reseptörler hematopoietik kök hücre homing katılıyor. [1, 2] Bu yazıda, hematopoietik kök hücre çalışmalarında kullanılan klasik homing protokol özetliyor. Genel olarak bu homing ihtiyaçları araştırılmalıdır hücre popülasyonu izole Bu nüfusun ilgi bir boya ile boyanması ve bu hücrelerin bir alıcı hayvan kan akışı içine enjekte içerir. Alıcı hayvan sonra enjeksiyon ve ilgi boya için pozitif hücrelerin yüzdesi ya da mutlak sayısı için değerlendirilen kemik iliği sonra önceden belirlenmiş bir zamanda kurban. En yaygın deneysel şemaları, genetik olarak farklı iki hayvanlardan elde edilen hematopoietik hücreleri (vahşi bir hayvan ve ilgili knock-out) homing verimlilik biri karşılaştırılır. Bu makale, deney gibi çerçevesinde hematopoetik hücre homing protokol tanımlamaktadır.

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

Bu makale, kemik iliği hematopoetik hücrelerin kendi nişler homing çalışma için kullanılan bir protokol tanımlamaktadır.

Kemik İliği Hematopoetik Hücreler, Homing

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

JoVE Journal

Biology

29.4K Görüntüleme.  MGH - Massachusetts General Hospital. Bu makale, kemik iliği hematopoetik hücrelerin kendi nişler homing çalışma için kullanılan bir protokol tanımlamaktadır.

Video: 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.

Plasenta Hematopoetik Kök Hücre İzolasyonu ve Analizi

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. ...

Biyoloji

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.

Myeloid dendritik hücreler kemik iliği öncüleri Kültür

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.

Kemik İliği Üretimi 2-foton Görüntüleme Kullanım murin Dendritik Hücreler Türetilmiş

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.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

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.

Lentiviral aracılı demonte sırasında Ex vivo İnsan Hematopoetik Kök Hücre Eritropoiesis

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.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

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.

Fare Kemik İliği osteoklast türetilmesi

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.

Kemik iliği türevi fare dendritik hücreleri ve makrofajlar floresan Fusion proteinlerin ifade

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.

Sıçan kemik Iliği Osteoclast precursors yalıtım, arıtma ve farklılaşma

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.

Fare Taze Kemik İliği Eksplantlarının Proplatelet Formasyon Dinamikleri

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