Diseksiyon ve izolasyon fare yolk kesesi ve aort-gonad-mesonephros bölgenin erken hematopoetik kök hücrelerin <ol><li> Başlamadan önce, prosedürü için gerekli tüm reaktifler ve araçlar için önemlidir: <ul><li> Makas </li><li> Forseps, büyüklüğü 4 ve cerrahi sınıf kıvrık uçlu forseps, büyüklüğü 5 / 45 (isteğe bağlı) </li><li> Şırınga ile 30 insülin iğnesi </li><li> 35mm ve 100mm Petri kapları </li><li> PBS% 10 ve% 20 fetal sığır serumu </li><li> ve son olarak, kollajenaz </li></ul></li><li> Diseksiyonu başlamak için, biz yazılan anlayışı 10.5 gün hamile bir kadın rahim boynuzları hasat gerekir. </li><li> Hayvanlar onaylanmış prosedürlere göre ötenazi. Biz, karbon dioksit boğulma, servikal dislokasyon kullanın. </li><li> Kadın karnı etanol ile ıslak ve cilt makas açıldı. </li><li> Forseps ile cilde Holding, periton ek kesikler açmak için yapılır. </li><li> Karın açılır ve rahim boynuzları görüntülenebilmekte. Uterin boynuz tutmak için forseps kullanarak, her distal uç ve iki boynuz tüketim merkezi kesti. </li><li> Uterus boynuzları sonra rahim dokuları temizlemek için% 10 FBS ile soğuk PBS içeren bir Petri kabındaki yerleştirilir. </li><li> Boyutu 4 forseps kullanarak ve mikroskop altında çalışan, yavaşça plasenta ponksiyon dikkatli olmak, her embriyo konseptus etrafında endometrial doku kaldırmak. </li><li> Kaldırılır sonra, embriyolar soğuk PBS ve% 10 FBS ile taze bir tabak içinde yerleştirilir. </li><li> Plasental doku embriyonun kendi noktada plasenta uzak embriyo kaplı yolk sac kesmek için forseps kullanarak yolk sac kaplı ortaya çıkarmak için dikkatlice çıkarın. Vitellin arterler, ve göbek kordonu bir kısmını içeren yolk kesesi embriyo hafifçe uzak alay ve disosiasyon için buz üzerinde bir kenara olabilir. </li><li> Embriyo% 10 FBS PBS minimal düzeyde küçük bir Petri kabı taşınır yeterli embriyo ıslak ama çok fazla değil. Plaka Diseksiyon sırasında yavaş yavaş, güvenli bir şekilde embriyo tutmak, ajite ise optimal düzeyde embriyo sabit tutmak. </li><li> Ön ayakları üstünde ve arka ayaklarının altında kesim tarafından embriyonun alt ve üst kısımları kaldırmak için kullanılan 30 gauge iğne, geçin. Kesme aletleri gibi iğneler kullanarak aşina olmak biraz pratik sürebilir. Iğneler geçerek tekrarlanan küçük kesimler kullanarak bir yöntem, baskın yapılan iğne baskın olmayan elle tutulur bir kesim kılavuzu olarak hareket etmek bir iğne ile doku disseke. </li><li> Embriyonun dorsal kısmını çıkarın. Bu Somitlerin içeren bir dokudur. Aorta (kırmızı çizgi) herhangi bir Knicks önlemek için yeterli bir mesafe kalarak en dorsal doku yavaşça kesip. Kesme aort, kan kaybı ve kolayca yerini görselleştirmek için yetersizlik neden olacaktır. Yine iğne ile küçük kesikler hassas kesim yeri korumak için hayati önem taşımaktadır. </li><li> Uzak görünümü dışında ekstra doku itin ve tekrar knicking olmadan, mümkün olduğunca aort yakın olarak kesme, ventral doku kesmek için embriyo turn around. Keser, en uygun yerden çok uzakta AGM izole bir kez silinebilir mevcut olan karın dokuları, neden olacaktır. </li><li> Embriyonun plaka karşı aşağı dorsal tarafına veya arkasına yerleştirin. Hafifçe yanlardan aşağı iterek ve gonadal sırtlar yer görselleştirme açık embriyo Şevli. Bunlar aortanın her iki tarafta tüp benzeri yapılar olarak off-center biraz bulunmaktadır. </li><li> Daha önce olduğu gibi aynı kesim teknikleri kullanarak, aşırı yan dokuları kaldırarak diseksiyonu tamamlayın. Embriyo yan dokusu çıkarıldıktan sonra, daha sonra embriyo kalan yan doku çıkarmak için baş aşağı açıktır. </li><li> Bu noktada, AGM, aort ya da gonadlar yaralanma olmadan iğne ile kolayca çıkarılabilecek olan herhangi bir aşırı doku denetlenir. Arıtma için sadece birkaç hücre yüzey belirteçleri güveniyor beri kök hücre popülasyonu daha iyi izolasyon olası sonuç olarak çok daha fazla olarak kaldırılıyor. </li><li> Kıvrık uçlu forseps kullanarak, yavaşça disosiasyon için hazır olana kadar soğuk PBS içinde% 10 FBS ile her AGM ve yer kadar kepçe. </li></ol> Bölünmeler ve FACS Analizi <ol><li> Ayrı tüplerde doku, ilk etapta sarısı keseler ve AGMs ayırmak. </li><li> Pelet dokular kısaca Spin ve% 10 FBS ile PBS atın. </li><li> Dokuların yeterince karşılamak için yeterli hacmi ile,% 20 FBS PBS içinde% 0.25 kollajenaz doku tekrar </li><li> 37 derece dokuların inkübe edin. 25-30 dakika, yumurta sarısı keseler AGMs ve 35-40mins. </li><li> Inkübasyon sonunda, dokular hafifçe yukarı pipet ve tek hücre süspansiyonları aşağı disosiasyon. </li><li> Dokuların aşırı PBS wi yıkayınth% 10 FBS, pelet% 10 FBS ile taze steril PBS içinde hücre ve tekrar süspansiyon haline getirin. </li><li> FACS analizi için, hücreler üreticinin tavsiyelerine göre lekeli. Biz flow sitometri gerçekleştirmek için BD Biosciences bir FACSAria kullanın. Embriyonik hücreler boyutu ve kırılganlık nedeniyle, sıralama diyafram, biz de laboratuvarda çalışma, yetişkin hematopoetik hücreler daha büyük bir çap için ayarlanır. Aynı şekilde, basınç yetişkin hematopoetik hücreler tarafından gerekli koşulları daha az olacak şekilde ayarlanır. Örneğin, FACSAria sıralama, meme büyüklüğü 100 mikron ve sıralama basıncı 30 psi olacaktır olacaktır. </li><li> Belirteçlerinin cKit, CD34 ve CD41 ifade üçlü pozitif hücrelerin kök hücre analizleri için, izole edilir ve yumurta sarısı kesesi ve çift pozitif (cKit, CD34) sıralanmış FACS üzerinden AGM izole edilmiştir. Tipik bir komplo bu gibi görünecektir: cKit pozitif hücreler CD34 ve CD41 aynı anda veya tek tek analiz edilir. Genellikle hücreler pozitif popülasyonlarının bir alt kümesi olarak bulunan kök hücre nüfusu ile her bir belirteç için pozitif bir tonlama gösterir. Bu araziler ortalama 100-300 hücreleri yolk kesesi ve başına AGM ile iyi bir hücre verimi elde etmiş olduğunu bize göstermektedir. </li></ol>

isolation of early hematopoietic stem cells from murine yolk sac and agm

Fare embriyo, erken hematopoez yolk kesesi ve aort-gonad-mesonephros bölge içeren birden fazla organlar, aynı anda oluşur. Bu bölgeler embriyoların kan sisteminin kurulması çok önemlidir ve fetal karaciğer bonemarrow yetişkin kök hücrelerin gelişmesi ve daha sonra kök hücrelerin nihai hareketi sağlar. Erken hematopoietik kök hücreler, bu organların akım sitometrik sıralayarak daha saf bir nüfusa elde etmek için embriyonun mikrodiseksiyon yoluyla izole edilebilir. Bu kök hücre popülasyonlarının fetal ve yetişkin kök hücre havuzu nasıl katkıda belirsizliğini koruyor. Ayrıca, laboratuvar erken kök hücreler işlevsel, fetal ve yetişkin hematopoetik kök hücreler nasıl farklı inceler. Ayrıca, laboratuvar türlü hematopoietik kök hücrelerin farklı popülasyonlar ve çeşitli genetik modelleri bağlamında işlevsel rolü test. Koloni testleri ve kemik iliği nakli: Bu videoda, biz, sık kullandığınız ve aynı zamanda bu nadir nüfusun izole tipik bir FACS plotfter sonuçlarını göstermek mikro diseksiyon prosedürü göstermektedir, fonksiyonel testlerinin de dahil olmak üzere çeşitli gerçekleştirmek mümkündür.

Watch this Scientific Journal Video about Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM at JoVE.com

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

Bu video yolk kesesi ve aort-gonad mesonephros bölge embriyolardan mikro incelemek ve hematopoietik kök hücreler sıralamak için flow sitometri kullanmak nasıl gösterir.

Murin Yolk Sac ve AGM Erken Hematopoetik Kök Hücre İzolasyonu

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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Research

JoVE Journal

Biology

14.6K Görüntüleme.  Brigham and Women's Hospital and Harvard Medical School. Bu video yolk kesesi ve aort-gonad mesonephros bölge embriyolardan mikro incelemek ve hematopoietik kök hücreler sıralamak için flow sitometri kullanmak nasıl gösterir.

Video: Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

İzolasyon ve Hematopoetik Kök Hücre Transplantasyonu (HKH&#39;lerin)

Biyoloji

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 ...

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. ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Mürin Hematopoetik Kök Hücreler ve Lineage-kararlı progenitörlerin Fenotipik Analizi ve İzolasyon

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Kemirgen Embriyonik retroviral Enfeksiyon Hematopoetik Farklılaşma Analizi için Hücre Türetilmiş embriyoidlerin Vücut Kök Hücreler

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Yetişkin Murine lacrimal ve submandibular bezlerden Myoepitelyal hücrelerin izolasyonu

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ geçici ve L-tipi kalsiyum akımının eşzamanlı ölçümleri için yüksek kaliteli murin atriyal ve ventriküler miyositlerin izolasyonu

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

Murin Kemik İliği Hematopoetik Kök ve Progenitör Hücrelerin ve Stromal Niş Hücrelerin Akış Sitometri Analizi

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs),&#160;defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

Uzun Süreli ve Kısa Süreli Hematopoetik Kök Hücreler için İzolasyon Yöntemi

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells ...