National Institutes of Health Kirchstein Bursu predoctoral dostluk AI-64.128 (MPM), GM-41.514 (MDC), GM-48.071 (IP)

1) bir fare hem de femur kemikleri çıkarın <ol><li> Kas kesip ve femur kemik ve eklemlerde aşağıda (diz ve kalça) üzerinde açığa çıkarmak için diseksiyon makas kullanın. Diseksiyonu cımbız ile femur merkezi tutun ve mümkün olduğunca sağlam epifiz kadar terk etmek üstünde ve eklemlerde aşağıda kesmek. </li><li> Küçük diseksiyon makas kullanılarak mümkün olduğu kadar çok kas olarak temizleyin. Femur RPMI bir tabak içine aktarın. </li><li> Tüm işlemler, bu noktada sadece steril ortam, araçlar, pipet uçları ve kültür yemekleri kullanarak Kaputun yürütülen olmalıdır. </li></ol> 2) femur kemikleri sterilize: <ol><li> Kaput, buz üzerinde% 70 etanol ile dolu küçük bir kültür çanak RPMI kemik transferi. </li><li> Kemikler için etanol içinde 5 dakika buz üzerinde emmek için izin verin. </li></ol> 3) Kemik iliği steril koşullar altında toplayın: <ol><li> Filtrelenmiş steril Temel DC kültür ortamı içeren küçük bir kültür çanak kemikleri aktarın. </li><li> Steril bir pens ile kemik atmak çanak veya tüp üzerinde tutun ve steril makas ile (kemik uçları) her epiphesis kesti. Cut kemik merkezi parlak kırmızı kemik iliği göstermelidir. </li><li> Steril bir şırınga ile (26-28 gauge iğne), 100-200 ml steril medya emmek. </li><li> Tutun steril medya taze bir yemek üzerine steril bir pens ile kemik ve kemik bir tarafına iğne yerleştirin. Kemiğinin üst kısmına yakın bir iğne ucu yerleştirin ve yavaş yavaş steril ortam ile kemik yıkayın. Kemik merkezi iseniz, iğne kolayca slayt gerekir. </li><li> Kemik iliği küçük parçalar halinde veya tek bir parça olarak, yıkar. Bu kemik ve steril medya çanak içine yıkanmalıdır. </li><li> Kemik iliği tamamen yıkamak gerektiği gibi, bu adımı tekrarlayın. </li><li> Kemik temiz, beyaz ve yarı saydam olacak. </li><li> Diğer femur kemik ile bu yordamı yineleyin. Temiz bir kez her femur kemik atın. </li></ol> 4) yeniden askıya alma ve Kemik iliği hücreleri aşağı Spin: <ol><li> Hücreler steril bir şahin tüpüne aktarın. </li><li> Iliği hala bozulmamış ise, çok nazik bir tek hücre süspansiyonu iliği break up, çanak ortamını pipet ve aşağı. Bu birkaç dakika sürebilir ve dik kuvvet tarafından hücreleri öldürerek önlemek için yavaş yavaş yapılmalıdır. </li><li> Herhangi bir diğer memeli hücrelerinde olduğu gibi Santrifüj hücreleri. </li></ol> 5) Hücre Lizis: <ol><li> Spin yapıldıktan sonra santrifüj tüpünü çıkarın ve medya kapalı (süzün) kaput küçük bir atık (50 ml şahin tüp) içine dökün. </li><li> Tüp yavaşça alt Flicking süspanse edin pelet. </li><li> Steril filtre edilmiş su ve 10x DPBS veya 10x PBS kullanarak, aşağıdaki hacimler kullanarak kırmızı kan hücreleri (eritrositler) kaldırmak için bir su lizis yürütmek: <ul><li> 900 ml steril filtre edilmiş su </li><li> 100 ml, 10x DPBS veya 10x PBS </li></ul></li><li> 10x PBS 5-10 saniye su ekledikten sonra 100 ml ekleyin. Sadece eritrositlerde parçalanmış olduğunu derhal Bunu yapmak çok önemlidir. Bu, hem pipetler hazır doldurulmuş ve iyi bir fikir. </li><li> - 5 ml, 10 ml steril DC Temel ortam ekleyin. </li></ol> 6) Hücreler Sayısı: <ol><li> Kaputun, tüp nazikçe hafif bir inversiyon, 45 derecelik bir eğim (medya tüp üst dokunur böylece) ile dönen 6 ila 11 ml kemik iliği hücreleri karıştırmak </li><li> Sayımı için steril bir tüp içine 10 ml medya çıkarın. </li><li> Re-cap hücreleri ve santrifüj tekrar. </li></ol> 7) Kaplama Dendritik hücreler: <ol><li> Hücreler 1 x 106/mL yoğunluğu kaplı olmalıdır. </li><li> İlköğretim DC Medya kaplama için uygun miktarda santrifüj hücreleri, süzün medya ve yeniden askıya alma hücreleri çıkarın. </li><li> Doku kültürü inkübatör yerleştirin. </li></ol> 8) Kültür Bakım ve Olgunlaşma: Her zaman daha fazla medya ekleyerek veya herhangi bir deney kullanarak önce kültürler kontrol edin. Hücre genel sağlık kontrol edin ve potansiyel kirlenme bakın. GÜN: 0 Dendritik hücreler kaplama. 3. GÜN: medya ve olmayan adherant hücrelerin% 75 çıkartın ve geri İlköğretim DC medya ekleyebilirsiniz. 6. GÜN: İlk Hücre Kaplama sonra aşağıdaki yordamı kullanarak yeniden plaka hücreleri: <ol><li> (Yapışık hücrelere kurumasına istemiyorum) çok hafif ıslak tabak bırakarak, 50 ml steril bir şahin tüp olmayan bazı adherant DC&#39;ler bu noktada, yeri ve içermelidir medya, Kaldır </li><li> Her plaka için PBS içinde 3 mM EDTA ekleyin ve 5 dakika oturmak için izin verir. </li><li> Plaka 45 derecelik bir açıyla tutun ve yavaşça olmayan adherant hücrelerin nazikçe çıkarmak için plakanın alt karşı medya pipet ve aşağı. </li><li> EDTA / PBS kalın gösteren hücrelerin alt toplanan ediliyor olması gerektiğiniplakası. </li><li> Bu birkaç dakika sonra, hücre karışımı 50 ml şahin tüpüne aktarılabilir. </li><li> Her plaka için bu işlemi gerçekleştirdikten sonra tüm hücreleri Spin. </li><li> Sayım ve plaka hücrelerin yoğunluğu 1 x 10 ^ 6 yeni steril 10 cm kültür kaplarına Orta DC Medya başına plaka. Hücreler doku kültürü inkübatör dönün. </li></ol> GÜN 10-11 Olgun DC&#39;ler stimülasyon / antijen yükleme için hazır. 9) Dendritik Hücre Uyarım: <ol><li> LPS (lipopolyscaccharide), aktivasyon ve peptid yükleme için istenen peptid ile birlikte kullanın. LPS kaynağına bağlı olarak, farklı stimülasyon koşulları daha iyi sonuçlar verebilir. Ayrıca, DC tanıtımı için peptid istenilen miktarda optimize edilmelidir. Bu protokol Uyarım koşulları 100 ng / ml LPS ve 100 ug / ml OVA (ovalbumin). </li><li> Yük antijen / hasattan önce 18-24 saat LPS ile tedavi /. </li></ol> Dendritik Hücre Medya: Steril filtre sonra her şeyi eklenmiştir Farklı dendritik hücre kültürü koşullarında bir inceleme ve dendritik hücre fenotipi için tartışma bölümüne bakınız. Bu kültür koşulları olgun dendritik hücreler üretmek için tasarlanmış hızla subkutan enjeksiyon ve verimli bir şekilde mevcut antijen sonra 18-24 saat drene lenf düğümleri, ev. İlköğretim DC Medya: Steril filtre sonra her şeyi eklenmiştir <ul><li> IMDM (500ml son hacmi 55-60 ml kaldırmak) 500 ml </li><li> 50 ml ısı inaktive FBS </li><li> 5 ml, 200 mM L-Gln, 2 mM nihai konsantrasyonu </li><li> 100 IU / ml penisilin ve 100 mg / ml streptomisin (10.000 IU / mL Kalem ve 10.000 mg / ml Strep stok 5 ml) </li><li> 50 UM B-Me (14.3 M B-Me stok, kimyasal kaput 1:100 sulandırmak, 50 mikromolar konsantrasyonu için 100 ml medya başına 35 ul kullanabilirsiniz) </li><li> 20-30 ng / ml GM-CSF </li><li> 100-400 IU / mL, IL-4, kabaca 10 - 40 ng / ml </li></ul> Olgunlaşma * SecondaryDC medya 100 ng / ml, TNF-alpha (Birincil DC medya eklemek) * Not: GM-CSF + IL-4 immatür dendritik hücreler üretmek gerekir. GM-CSF kullanımı + IL4 (400 IU / ul) + TNF-alpha (100 ug / ml), olgun monosit kökenli dendritik hücreler benzer hücreler yaratacaktır. Olgun dendritik hücrelerin immatür dendritik hücreler ve kültürler endojen TNF-a stromal hücreler yayımlanan 4 olabilir gibi verimli antijen almamaya bilinmektedir.

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Dendritik hücreler, adaptif immün yanıt, bugüne kadar karakterize en etkili antijen sunan hücre anahtar arabulucular. Literatürde insan ve fare dendritik hücre kültürü yöntemleri farklı dendritik hücre tiplerinin gelişimini etkilemek için kullanılan sitokinlerin türü farklıdır. Özellikle, GM-CSF, flt3L, IL4, IL13, TNF-alfa ve IFN-gama, olgun, olgunlaşmamış, inflamatuvar ve kararlı durum in vitro 3-7 dendritik hücreler elde edilen fare kemik iliği gibi üretmek için farklı kombinasyonları kullanılır...

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

Fare dendritik hücrelerin hazırlanması için çeşitli yöntemler literatürde bulunabilir. Burada, kültürde% 85 daha fazla CD11c yüksek dendritik hücreler üreten bir yöntem mevcut subkutan enjeksiyon ve antijen spesifik T hücreleri (video) mevcut antijen sonra drenaj lenf nodu ev. Ayrıca, 10 gün, kültürlü hücrelerinin morfolojisi, olgun bir dendritik hücre ve hücre &lt;% 85 CD11chigh olan tipik, dendritik hücre olgunlaşması izlemek Essen Aletleri Incucyte kullanın. 2-foton görüntüleme ile periferik lenf düğümlerine antijen sunumu çalışma dendritik hücre ve T hücre etkileşimi 1, 2, üç farklı aşama olduğunu ortaya çıkardı. Faz I, son derece hareketli bir antijen spesifik T hücreleri ve dendritik hücreler 1, 2 taşıyan antijen arasında kısa seri kişilerin oluşur. Faz iki antijen spesifik T hücre ve dendritik hücreler 1, 2 yatak antijen arasındaki uzun süreli temas ile işaretlenir . Son olarak, Faz III, dendritik hücreler ayrılmakta motilite kazanmak ve 1, 2 bölmek için, T hücreleri tarafından karakterize edilir . Bu antijen yüklü hücre izci boya etiketli dendritik hücreler iki-foton görüntüleme ile analiz edilebilir antijen-spesifik etkileşimler türünün tek örneğidir.

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

Dendritik hücreler tarafından ikincil lenfoid organlara Antijen sunumu T hücre aracılı adaptif immün yanıt başlatılması için çok önemlidir. Burada kemik iliği kültürü elde edilen fare dendritik hücreler, aktivasyon, 2-foton görüntüleme için etiketleme göstermektedir.

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% ...

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

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JoVE Journal

Biology

31.0K Görüntüleme.  University of California, Irvine (UCI). Dendritik hücreler tarafından ikincil lenfoid organlara Antijen sunumu T hücre aracılı adaptif immün yanıt başlatılması için çok önemlidir. Burada kemik iliği kültürü elde edilen fare dendritik hücreler, aktivasyon, 2-foton görüntüleme için etiketleme göstermektedir.

Video: 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.

Farelerde Periferik lenf nodu diseksiyonu ve 2-Foton Görüntüleme

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

Biyoloji

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.

2-Foton Mikroskop kullanarak Fare Thymus in situ Görüntüleme

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.

Kranial Pencere üzerinden neokorteks Kan Akış 2-Foton Görüntüleme Bir Yöntem

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 .

Katman In Vivo 2-Foton Kalsiyum Görüntüleme Fare 2 / 3

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.

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 ...

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 ...

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.

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 ...

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 ...