Femur ve süresince tibialar itibaren Kemik İliği Hasat <ol><li> 12-haftalık fare (BALB / c farelerde kullanarak ancak herhangi bir fare suşu ile bu işlemi yapabilirsiniz) - 6 femur ve süresince tibialar kemik iliği hasat prosedürün ilk adımı. Başlamak için, CO 2 inhalasyon (gösterilmeyecektir) fare euthanize. Hasat femur ve her birinin arka ayakları süresince tibialar. Sonra, bir tıraş bıçağı ile, kalça ve diz eklemleri kaldırmak için her iki ucunda femur kesmek ve kemik iliği maruz. </li><li> Benzer bir şekilde, diz ve ayak bileği komşu bölgeleri kaldırmak ve süresince tibialar iliği açığa çıkarmak için her iki ucundaki kesti. </li><li> 26-gauge iğne ile 3 ml şırınga alın ve RPMI takviye ile% 10 FBS, penisilin, streptomisin ve beta-mercaptoethanol (BME = 100 um) ile doldurun. Cımbız kullanarak, kemik RPMI medya içeren Petri üzerinde tutun. Şırınga iğnesi kemiğin bir ucunu takın ve kemik iliği dışarı atılması için piston bastırın. Şırınga iğnesi artık iliği dışarı floş kemik içinde yukarı ve aşağı hareket ettirilebilir. </li><li> Tüm kemikler için prosedürü tekrarlayın ve sonra kültürler kurulması ile devam. </li></ol> Kültürler kurulması <ol><li> Kemik iliği kültürleri kurmak için, bir hücre süspansiyonu doku kırmak için birkaç kez aşağı kızarmış kemik iliği pipet ve. </li><li> 50 ml konik tüpün üstüne bir 70 mikron filtre koyun ve hücre süspansiyonu filtre ekleyin. </li><li> Petri kabı RPMI ile bir kez yıkayın, sonra 70 mikron filtre durulama ekleyin. </li><li> 1 ml şırınga pistonu kauçuk ucunu kullanarak, 70 mikron filtre üstünde kalan kemik iliği adet eziyet. </li><li> RPMI ile filtre bir kez yıkayın. </li><li> Filtre yıkadıktan sonra, hücre süspansiyonu için 1200 rpm&#39;de 5 dakika santrifüj. </li><li> PBS içinde resuspending hücre pelletini yıkayın. </li><li> Hemasitometre kullanarak hücrelerin güvenin. Biz genellikle bir fare kemikleri (iki femurları artı iki süresince tibialar) ~ 90 milyon hücre edinin. Hücrelerinin sayımı sonra, onları tekrar 1200 rpm&#39;de 5 dakika santrifüj. </li><li> RPMI ve kısım son konsantrasyon ml başına 1 milyon hücre olduğunu yeterince RPMI içeren doku kültürü şişeleri içine küçük bir miktar hücrelerin tekrar </li><li> Ilgi hücre tipi kalkınmayı teşvik etmek sitokinler ekleyin. Bu durumda, sitokin interlökin-3 (IL-3 = 10 ng / ml), bazofil ve mast hücrelerinin gelişimini teşvik etmek eklenir. Bazofiller edinmek için, 10 gün boyunca kuluçkaya yatmaktadır. Mast hücreleri için, 5 hafta boyunca kuluçkaya yatmaktadır. Mast hücreleri oluştururken, medya ve IL-3 haftada bir kez değişmiş olmalıdır. </li><li> Burada mast hücreleri sadece kaplama sonra nasıl görünmelidir bir bakış. Bu görüntü, bazofil ve mast hücreleri interlökin-3 ilave edildikten sonra 10 gün mast iliği hücreleri farklılaşma göstermektedir. </li><li> İstenen hücre tipi bir kere elde edildiğinde, elektroporasyon adım ile devam edin. </li></ol> Electroporating Hücreler <ol><li> Elektroporasyon adım başlamak için, kültür şişelerinde hücrelerinin sayısını belirlemek. </li><li> Hücreler genellikle mL başına 10 milyon bir yoğunluk electroporated, bu yüzden konik tüpler için gerekli hücre sayısı transfer tüpler ve 1200 rpm&#39;de 5 dakika santrifüj. </li><li> Santrifüj sonra, medya aspirat, 1X PBS ile hücreleri yıkayın ve tekrar 1200 rpm&#39;de 5 dakika santrifüj. </li><li> PBS aspire ml başına 10 milyon hücre hücre süspansiyonu yapmak için Bio-Rad Gene Verici elektroporasyon tampon ekleyin. </li><li> Hücrelerin resuspending sonra, son bir konsantrasyonda ml başına 10 ila 20 mikrogram istenen plazmid DNA ekleyin. </li><li> 96-iyi elektroporasyon plaka üzerinde seçtiğiniz kuyu içine hücre süspansiyonu kısım 150 mikrolitre. </li><li> MXcell plaka odasında elektroporasyon plaka koyun ve kapağını kapatın. </li><li> Mast hücrelerinin transfeksiyon önce bir ön ayar veya saklanan bir protokol kullanarak sürece, elektroporasyon protokol MXcell içine programlanmış olmalıdır. Optimizasyonu deneyler sayesinde mast hücrelerinin yüksek transfeksiyon verimliliği kare dalga darbe protokolleri kullanarak ortaya keşfettiler. Biz 2000uF ve 1000 ohm 300V/20ms, 350/15ms, 350V/20ms ve 350V/10ms kare dalga darbeleri teslim plaka üzerinde elektroporasyon koşulları değişecektir. Protokol ve cihaz üzerine yüklendikten sonra, hücrelerin electroporate &quot;Darbe&quot; tuşuna basın. </li><li> Elektroporasyon tamamlandıktan sonra, bir doku kültürü plakası hücrelere aktarın. Biz genellikle interlökin-3 ml başına 10 nanogram 300 mikrolitre RPMI (% 10 FCS, penisilin, streptomisin ve beta-mercaptoethanol ile desteklenmiş) içeren bir 48-iyi doku kültürü plakası sıra her 150 mikrolitrelik elektroporasyon örnek aktarın. Hücreler 37 gece inkübe ° C, daha sonra ifade ve bastırılması için 24 saat sonra çalışılmalıdır. </li></ol> Transfection Sonuçlar Plazmid ml GFP başına 20 mikrogram kullanarak başarılı elektroporasyon sonra hücreler florasan mikroskobu görüntüsü, video gösterilir. Yaklaşık% 30 MXcell elektroporasyon sistem transfeksiyon verimliliği kullanılarak elde edilebilir. Sistem, hücre canlılığını korurken, transfeksiyon verimliliğini en üst düzeye çıkarmak için şartlarda değişiklik sağlar.

Daha genomik bilgi ortaya çıkıyor ve böyle bir siRNA olarak yeni araçlar geliştirilmiştir, fizyolojik ilgili hücrelerin kullanımı, hastalık yolları, protein-protein etkileşimleri, ve sinyal iletimi anlayışımızı ilerletmek için her zamankinden daha önemli hale gelmiştir. İlköğretim hücreleri, dokuları veya sıvıları doğrudan elde edilen ve in vitro olarak yetiştirilmektedir. Bu hücreler, genetik materyalin eksojen tanıtım yoluyla yolları da dahil, bir dizi manipüle edilebilir. Bu plazmid DNA veya birincil hücre içine siRNA tanıtmak için en etkili yolu elektroporasyon olduğunu giderek daha belirgin hale gelmektedir. Elektroporasyon geçici böylece ekzojen nükleik asitlerin hücre girmesine izin verir, hücre zarının geçirgenliğini artırmak amacıyla elektrik darbeleri hücreleri ortaya koyar. Transfeksiyon Bu yöntem, farklı hücre türleri / hatları ve, böylece mast hücrelerinin transfect kullanılabilir yanı sıra, diğer kemik iliği kaynaklı hücre arasındaki farklılıkların karşılamak için optimize edilebilir. Ayrıca, viral aracılı transfeksiyon aksine, elektroporasyon transfekte DNA boyutu sınırlar empoze, ne de kapsamlı bir hazırlık gerektirir değildir. Elektroporasyon lipid aracılı transfeksiyon aksine, daha az toksik olabilir ve transfekte nükleik asit endosomal yakalama sonuç vermez. Bio-Rad, bu hücrelerin MXcell elektroporasyon sistemi için özel olarak geliştirilmiştir. MXcell transfeksiyon verimliliği ve hücre canlılığı en üst düzeye çıkarmak için şartlarda değişiklik sağlar. Transfeksiyon verimliliği optimize etmek ve hücre ölümü, voltaj elektroporasyon parametreleri de dahil olmak üzere bir çok en aza indirmek için, kondansatör, direnç ve nabız uzunluğu ayarlanabilir ve değerlendirilmelidir.

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<td>Gene Pulser MXcell Electroporation System</td>
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<td>Gene Pulser Electroporation Buffer</td>
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the preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation

Elektroporasyon birincil hücre içine plazmid DNA veya siRNA tanıtmak için en etkili yolu olduğu giderek daha belirgin hale gelmektedir. Gene Verici MXcell elektroporasyon sistemi ve Gen Verici elektroporasyon tampon, özellikle memeli hücrelerinde ve birincil ve kök cells.This video gibi zor-transfect hücreleri, nükleik asitlerin transfect geliştirilmiştir birincil fare kemik iliği hematopoetik hücre kültürleri nasıl kurulacağını gösteren ve sonra MXcell sistemi elektroporasyon için hazırlamak. Biz femur ve tibia izole ederek başlar. Femur ve tibia hem de kemik iliği biçilir ve kültürler kurulur. Kültüre kemik iliği hücreleri daha sonra transfekte ve analiz edilir.

Watch this Scientific Journal Video about The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation at JoVE.com

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

Bu prosedür, birincil fare kemik iliği hematopoetik hücre kültürleri kurmak ve transfeksiyon Gene Verici MXCell elektroporasyon sistemini kullanarak takip nasıl açıklamaktadır.

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 ...

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Biology

25.6K Görüntüleme.  Bio-Rad Laboratories, Inc.  ChineseVersion 

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

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Biyoloji

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Geç Stage Drosophila pupa Brain İlköğretim Nöronal Kültürler

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Mikobakteriler, Elektroporasyon

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Kemirgen Yenidoğanlar İlköğretim grubuyla orta beyin dopamin Hücre Kültürleri

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

İlköğretim Kültür ve Corti murin Organ Plazmid Elektroporasyon.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Birincil Hücre Kültürleri Drosophila Gastrula Embriyolar

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Bazal Omurgalı soylarından İlköğretim andMyogenic Öncü Hücre / Miyoblast Kültürlerinin Hazırlanması

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

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 ...