Bu çalışma, Amerikan Kalp Derneği (ÇED 0740129N) ve NIH T32 HL90610 tarafından desteklenmiştir.

<ol>
	<li>Goodwill, A. G., Frisbee, S. J., Stapleton, P. A., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Chronic+Anticholesterol+Therapy+on+Development+of+Microvascular+Rarefaction+in+the+Metabolic+Syndrome.">Impact of Chronic Anticholesterol Therapy on Development of Microvascular Rarefaction in the Metabolic Syndrome.</a> Microcirculation. , 1-18 (2009).</li><li>Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vascular+thromboxane+generation+impairs+dilation+of+skeletal+muscle+arterioles+of+obese+Zucker+rats+with+reduced+oxygen+tension.">Increased vascular thromboxane generation impairs dilation of skeletal muscle arterioles of obese Zucker rats with reduced oxygen tension.</a> Am. J. Physiol. Heart Circ. Physiol. 295, H1522-H1528 (2008).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-dependent+changes+in+endothelial+factors+regulating+arteriolar+tone.">Growth-dependent changes in endothelial factors regulating arteriolar tone.</a> Am. J. Physiol. Heart Circ. Physiol. 292, H207-H214 (2007).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+emerges+as+a+regulator+of+tone+in+skeletal+muscle+arterioles+during+juvenile+growth.">Hydrogen peroxide emerges as a regulator of tone in skeletal muscle arterioles during juvenile growth.</a> Microcirculation. 15, 151-161 (2008).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+myogenic+responsiveness+of+skeletal+muscle+arterioles+with+juvenile+growth.">Increased myogenic responsiveness of skeletal muscle arterioles with juvenile growth.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 2344-2351 (2008).</li><li>Dacey, R. G., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+rat+intracerebral+arterioles:+methods,+morphology,+and+reactivity.">A study of rat intracerebral arterioles: methods, morphology, and reactivity.</a> Am. J. Physiol. Heart Circ. Physiol. 243, H598-H606 (1982).</li><li>Fredricks, K. T., Liu, Y., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+extraparenchymal+resistance+arteries+of+rat+skeletal+muscle+to+reduce+PO2.">Response of extraparenchymal resistance arteries of rat skeletal muscle to reduce PO2.</a> Am. J. Physiol. 267, H706-H715 (1994).</li><li>Durand, M. J., Raffai, G., Weinberg, B. D., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-(1-7)+and+low-dose+angiotensin+II+infusion+reverse+salt-induced+endothelial+dysfunction+via+different+mechanisms+in+rat+middle+cerebral+arteries.">Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299, H1024-H1033 (2010).</li><li>LeBlanc, A. J., Cumpston, J. L., Chen, B. T., Frazer, D., Castranova, V., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+inhalation+impairs+endothelium-dependent+vasodilation+in+subepicardial+arterioles.">Nanoparticle inhalation impairs endothelium-dependent vasodilation in subepicardial arterioles.</a> J. Toxicol. Environ. Health A. 72, 1576-1584 (2009).</li><li>Jernigan, N. L., LaMarca, B., Speed, J., Galmiche, L., Granger, J. P., Drummond, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+salt+enhances+benzamil-sensitive+component+of+myogenic+constriction+in+mesenteric+arteries.">Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H409-H420 (2008).</li><li>Stapleton, P. A., Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mechanisms+of+endothelium-dependent+dilation+in+skeletal+muscle+arterioles+with+genetic+hypercholesterolemia.">Altered mechanisms of endothelium-dependent dilation in skeletal muscle arterioles with genetic hypercholesterolemia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1110-R1119 (2007).</li><li>Goodwill, A. G., Stapleton, P. A., James, M. E., d'Audiffret, A. C., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+arachidonic+acid-induced+thromboxane+generation+impairs+skeletal+muscle+arteriolar+dilation+with+genetic+dyslipidemia.">Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia.</a> Microcirculation. 15, 621-631 (2008).</li><li>Baumbach, G. L., Hadju, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+composition+of+cerebral+arterioles+in+renal+and+spontaneously+hypertensive+rats.">Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.</a> Hypertension. 21, 816-826 (1993).</li><li>Uchida, E., Bohr, D. F., Hoobler, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+studying+isolated+resistance+vessel+from+rabbit+mesentery+and+brain+and+their+responses+to+drugs.">A method for studying isolated resistance vessel from rabbit mesentery and brain and their responses to drugs.</a> Circ. Res. 4, 525-536 (1967).</li><li>Davis, M. J., Kuo, L., Chilian, W. M., Muller, J. M. I. s. o. l. a. t. e. d., Barker, J. H., Anderson, G. L., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> Isolated, perfused microvessels. In: Clinically Applied Microcirculation Research. 32, 435-456 (1995).</li><li>Lombard, J. H., Liu, Y., Fredricks, K. T., Bizub, D. M., Roman, R. J., Rusch, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+rat+middle+cerebral+arterieal+to+reduced+PO2+and+prostacyclin.">Electrical and mechanical responses of rat middle cerebral arterieal to reduced PO2 and prostacyclin.</a> Am. J. Physiol. 276, H509-H516 (1994).</li></ol>

Çıkar çatışması ilan etti.

Bu genel teknik kolaylıkla en dokular için uygulanabilir olmasına rağmen sunulan protokolü, bir iskelet kası mikrodamar izolasyonu, çıkarma ve çift kanül açıklanmaktadır. Mevcut yazı için dönem &quot;arteriyol&quot; da önemli bir bir organ perfüzyon direnç düzenlemesine katkı sağlayan veya dinlenen aktif ton altında çapı 70-120 mikron arasında değişen bir direnç damarı tanımlamak için yazarlar tarafından kullanılmıştır doku. Bazı değişikliklerle, bu si...

<table border="1"><tbody><tr><td> Reaktifler ve Ekipmanları </td><td> Şirket </td><td> Yorumlar / Katalog # </td></tr><tr><td> Gemi Odası </td><td> Özel </td><td> Dave Eick (MCW) </td></tr><tr><td> Isıtmalı Sirkülasyon Su Banyosu </td><td> PolyScience ve Haake </td><td> Haake DC 10 </td></tr><tr><td> Pipetleri </td><td> Frederick Haer &amp; Co </td><td> Kılcal Boru 2,0 mm OD x 1.0 mm ID (27-33-1) </td></tr><tr><td> Basınç Monitörü </td><td> Dünya Hassas Aletler </td><td></td></tr><tr><td> Su Ceketli Rezervuar </td><td> Özel </td><td></td></tr><tr><td> Dış Işık Kaynağı </td><td> Dünya Hassas Aletler </td><td> Novaflex </td></tr><tr><td> Pipetleyin Çektirme </td><td> Mikro veri Aletleri </td><td> PMP102 mikropipet Çektirme </td></tr><tr><td> Tam Complecerrahi aletler Ment </td><td> Güzel Bilim Araçları </td><td> Dumont </td></tr><tr><td> Ultra İnce Forseps </td><td> Güzel Bilim Araçları </td><td> Inox # 5 </td></tr><tr><td> İpek Sütür Konu </td><td> Ethilon </td><td> # 10-0 veya 9-0 </td></tr><tr><td> Stereo Mikroskop </td><td> Olympus </td><td> Olympus SZ-11 </td></tr><tr><td> Analog Video Kumpaslar </td><td> Boeckeler </td><td> Via Kontrolörü (Via-100) </td></tr><tr><td> Yüksek Çözünürlüklü Analog Kamera </td><td> Panasonic </td><td> GP-MF 602 </td></tr><tr><td> Oksijen Tankı </td><td> Bölgesel </td><td> % 21 denge azot ve% 5 CO 2 dengesi azot </td></tr><tr><td> Borular </td><td> Tygon </td><td></td></tr><tr><td> Pompa boşaltın </td><td> Cole Parmer Instrument Co </td><td></td></tr><tr><td> Modifiye Rat PSS </td><td> Aşağıda tarifi bak </td><td></td&gt; </tr><tr><td> Van Breemen en Relaxant PSS </td><td> Aşağıda tarifi bak </td><td></td></tr></tbody></table> Tablo 1. Rakamlar sunulan izole mikrodamar istasyonu kurulumu en önemli bileşenlerinden bir listesi. <table border="1"><tbody><tr><td> Modifiye Rat PSS tarifi </td><td> PSS iki litre yapmak için </td><td> 20X Tuz Stok (2L) </td><td> 20X Tampon Stok (2L) </td></tr><tr><td> NaCl </td><td></td><td> 278.0 g </td><td></td></tr><tr><td> KCl </td><td></td><td> 14.0 g </td><td></td></tr><tr><td> MgSO 4-7H 2 O </td><td></td><td> 11.5 g </td><td></td></tr><tr><td> CaCI2-H2O </td><td></td><td> 9.4 g </td><td></td></tr><tr> &lt;td&gt; NaHCO 3 </td><td></td><td></td><td> 80.8 g </td></tr><tr><td> EDTA </td><td></td><td></td><td> 0.4 g </td></tr><tr><td> NaH 2 PO 4 </td><td> 0.28 g </td><td></td><td></td></tr><tr><td> Glikoz </td><td> 1.98 g </td><td></td><td></td></tr><tr><td> 20x Tuz Stok </td><td> 100 mL </td><td></td><td></td></tr><tr><td> 20x Tampon Stok </td><td> 100 mL </td><td></td><td></td></tr><tr><td> Distile Su </td><td> 1800 mL </td><td></td><td></td></tr></tbody></table> Tablo 2. Izole mikrodamar protokolleri kullanılan standart fizyolojik tuz çözeltisi (PSS) için tarifi. Tarif Hakkında Yorum: Tuz Stok 2 L ve Tampon Stok 2 L olun. Bunlar cBir kullanılmadığı zaman buzdolabında, ama iyi ve çoğu PSS hazırlarken önce onları sallayın. Ilave maddeler nihai PSS hazırlanması sırasında eklendi. <table border="1"><tbody><tr><td> Van Breemen en Relaxant PSS </td><td> PSS 2 litre yapmak için </td><td> 20X Tuz Stok (1L) </td><td> 20X Tampon Stok (1L) </td></tr><tr><td> NaCl </td><td></td><td> 107.4 g </td><td></td></tr><tr><td> KCl </td><td></td><td> 7.0 g </td><td></td></tr><tr><td> MgSO 4-7H 2 O </td><td></td><td> 5.76 g </td><td></td></tr><tr><td> MgCl 2-6H 2 O </td><td></td><td> 81.32 g </td><td></td></tr><tr><td> NaHCO 3 </td><td></td><td></td><td> 40.4 g </td></tr><tr><td> EDTA </td><td></td><td></td><td> 0.2 g </td></tr><tr><td> EGTA </td><td></td><td></td><td> 15.22 </td></tr><tr><td> NaH 2 PO 4 </td><td> 0.28 g </td><td></td><td></td></tr><tr><td> Glikoz </td><td> 1.98 g </td><td></td><td></td></tr><tr><td> 20x Tuz Stok </td><td> 100 mL </td><td></td><td></td></tr><tr><td> 20x Tampon Stok </td><td> 100 mL </td><td></td><td></td></tr><tr><td> Distile Su </td><td> 1800 mL </td><td></td><td></td></tr></tbody></table> Tablo 3. Sıfır aktif ton arasında koşullar altında izole mikrodamar protokoller kullanılır Van Breemen en gevşetici fizyolojik tuz çözeltisi (PSS) için tarifi. Tarif Comments on: Bir Tuz Stok 1 L olunTampon Stok nd 1 L. Bunlar kullanılmadığı zaman buzdolabında, ama iyi ve çoğu PSS hazırlamadan önce onları sarsamaz. Ilave maddeler nihai gevşetici PSS hazırlanması sırasında eklendi.

the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity

İzole mikrodamar hazırlanması, böylece perfüzyon direnci 1-5 farklı faktöre katkı kontrol eden damar çapı incelenmesi için izin veren bir ex vivo hazırlanması, ve. Bu başlangıçta. 15 birkaç yıl önce Uchida ve ark, büyük ölçüde, olduğu klasik bir deney preparatıdır. Bu ilk açıklama kapsamlı öncelikle Virginia 6-8 Üniversitesi&#39;nde Dr Brian Duling laboratuvarında, değiştirilmiş ve geliştirilmiş oldu teknikleri için temel oluşturmuştur ve biz sayfalarda güncel bir yaklaşımla sunmak. Bu preparasyon özellikle tercih mikrodamar gibi bir sıçan gracilis arteriyol bakınız, ancak bazik hazırlanması kolay türler arasında yaklaşık 9-13 başka doku veya organ izole damarlarının uygulanabilir. Izole mikrodamarlar Mekanik (yani boyutlu) değişiklikleri kolayca değerlendirilebilirfizyolojik (örneğin, hipoksi, damar içi basınç veya kesme) veya farmakolojik zorluklar ve sağlam bir entegre yanıtları oluşan mekanik elemanlar içgörü sağlayabilir, ancak ex vivo, doku geniş bir dizi yanıt olarak. Bu yöntemin önemi de damar içi basınç (miyojenik), otonomik innervasyonu, hemodinamik de dahil olmak üzere, diğer kaynaklardan gelen katkıların çoğunun kontrolü için izin verirken o, mikrodamar çapında entegre regülasyonu üzerine etkilerinin kolay manipülasyon için izin vermesidir ( makaslama stresi), endotel bağımlı veya bağımsız uyaranlar, hormonal ve parankimal etkileri, kısmi bir liste sunmak için. Uygun deneysel koşullar altında ve uygun hedefleri ile, bu in vivo ya da kolayca geniş sistemik değişkenlerin uyduruk kontrolü için izin vermez yerinde doku / organ hazırlıklarda, üzerinde bir avantaj olarak hizmet verebilir. MaBu hazırlığın jor sınırlama aslında onun güçlü yönlerinden sonucudur. Tanım olarak, bu gemilerin davranışları vasküler rezistans düzenlenmesinde en önemli katkıda bulunan birçok Gibi, nöral humoral, metabolik, vb dahil olmak üzere, kaldırılmış koşullarda çalışılmaktadır, araştırmacı önlemek için aşırı ihtar verilir yorumlanması ve bu hazırlık kullanılarak toplanan verilerin ekstrapolasyonu. Bu hazırlık ile ilgili endişe diğer önemli alan bu tür endotelinde veya damar düz kas gibi hücresel bileşenlere zarar çok kolay olabilir yani, hata bu tür değişken kaynak sokulabilir. Kuvvetle bireysel araştırmacı deney başlangıcında ve periyodik bir protokol boyunca hem hazırlık kalitesini sağlamak için uygun ölçümler kullanmak önerilir.

Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

Bir<em&gt; Ex vivo</em&gt; Hazırlık vasküler vazoaktif uyaranlara tepkiler ve pasif duvar mekaniği ile temel yapısal özelliklerinin değerlendirilmesi hem de sorgulama için büyük gracilis kas direnci arteriol izolasyonu için açıklanmıştır.

Ex vivo</emVasküler Reaktivite incelenmesi için> İzole İskelet mikrodamar Hazırlık

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

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Research

JoVE Journal

Biology

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

10.6K Görüntüleme.  West Virginia University. Bir Ex vivo Hazırlık vasküler vazoaktif uyaranlara tepkiler ve pasif duvar mekaniği ile temel yapısal özelliklerinin değerlendirilmesi hem de sorgulama için büyük gracilis kas direnci arteriol izolasyonu için açıklanmıştır.

Video: The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

İskelet kas-içi hayati Mikroskopi in vivo Mikro sirkülasyon Ölçümü

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Biyoloji

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

İzole İskelet Kaslar Kasılma, çabuk yorulma ve Alternans Ex Vivo Değerlendirilmesi

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Yeni Bir<em&gt; Ex vivo</emEmbriyonik Fare Kalp için&gt; Kültür Yöntemi

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

İzole Kan-perfüze Sıçan Akciğer Hazırlık Tek Mikrodamar Geçirgenliği miktarının

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Farelerde Kardiyak inotropiyi Soruşturma İzole papiller Kaslar kontraktilite Ölçümleri

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

c-FOS Protein Immünohistolojik Algılama: Belirli Fizyolojik Yanıtları ilgilendim Merkez Pathways bir Marker Olarak Faydalı Aracı<em&gt; İn Vivo</em&gt; ve<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Ex vivo İzole İskelet mikrodamar Hazırlık