Name: proplatelet formation dynamics of mouse fresh bone marrow explants
Uploaded: 2021-05-20T15:00:00
Description: Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Yazarlar jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert'e teknik yardım için teşekkür etmek istiyor. Bu çalışma ANR (Agence National de la Recherche) Grant ANR-17-CE14-0001-01 ve ANR-18-CE14-0037 tarafından desteklenmiştir.

Tüm hayvan deneyleri Avrupa standartlarına uygun olarak gerçekleştirildi 2010/63/EU ve CREMEAS Strazburg Üniversitesi Hayvan Deneyleri Etik Komitesi (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg).
1. Reaktiflerin hazırlanması
<ol><li>Tablo 1'de açıklandığı gibi reaktifleri hazırlayın.<ol>
<li>Stok I için her tozu ayrı ayrı çözün. Preparatın osmolaritesinin 295 mOsm/L'den yüksek olduğundan emin olun. Bu çözelti bir yı...

<ol>
	<li>Thiery, J. P., Bessis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+platelet+genesis;+in+vitro+study+by+cinemicrophotography.">Mechanism of platelet genesis; in vitro study by cinemicrophotography.</a> Reviews in Hematology. 11 (2), 162-174 (1956).</li><li>Becker, R. P., De Bruyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transmural+passage+of+blood+cells+into+myeloid+sinusoids+and+the+entry+of+platelets+into+the+sinusoidal+circulation:+A+scanning+electron+microscopic+investigation.">The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into the sinusoidal circulation: A scanning electron microscopic investigation.</a> American Journal of Anatomy. 145 (2), 183-205 (1976).</li><li>Muto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scanning+and+transmission+electron+microscopic+study+on+rat+bone+marrow+sinuses+and+transmural+migration+of+blood+cells.">A scanning and transmission electron microscopic study on rat bone marrow sinuses and transmural migration of blood cells.</a> Archivum Histologicum Japonicum. 39 (1), 51-66 (1976).</li><li>Cramer, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+platelet+formation+by+human+megakaryocytes+cultured+with+the+Mpl+ligand.">Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.</a> Blood. 89 (7), 2336-2346 (1997).</li><li>Kaushansky, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+megakaryocyte+progenitor+expansion+and+differentiation+by+the+c-Mpl+ligand+thrombopoietin.">Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.</a> Nature. 369 (6481), 568-571 (1994).</li><li>Strassel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors.">Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.</a> Experimental Cell Research. 318 (1), 25-32 (2012).</li><li>Aguilar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+environmental+stiffness+for+megakaryocyte+differentiation+and+proplatelet+formation.">Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.</a> Blood. 128 (16), 2022-2032 (2016).</li><li>Scandola, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electron+microscopy+to+study+megakaryocytes.">Use of electron microscopy to study megakaryocytes.</a> Platelets. 31 (5), 589-598 (2020).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+megakaryocyte+development+in+the+native+bone+marrow+environment.">Characterization of megakaryocyte development in the native bone marrow environment.</a> Methods in Molecular Biology. 788, 175-192 (2012).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+megakaryocyte+morphology+and+proplatelet+formation+in+mice+with+megakaryocyte-restricted+MYH9+inactivation.">Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation.</a> Blood. 113 (14), 3182-3189 (2009).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proplatelet+formation+deficit+and+megakaryocyte+death+contribute+to+thrombocytopenia+in+Myh9+knockout+mice.">Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice.</a> Journal of Thrombosis and Haemostasis. 8 (10), 2243-2251 (2010).</li><li>Ortiz-Rivero, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C3G,+through+its+GEF+activity,+induces+megakaryocytic+differentiation+and+proplatelet+formation.">C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.</a> Cell Communication and Signaling. 16 (1), 101 (2018).</li><li>Junt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317 (5845), 1767-1770 (2007).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+MYH9-related+disease:+mutations+in+nonmuscle+myosin+II-A.">Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.</a> Blood. 119 (1), 238-250 (2012).</li><li>Malara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+structure+and+nano-mechanics+determine+megakaryocyte+function.">Extracellular matrix structure and nano-mechanics determine megakaryocyte function.</a> Blood. 118 (16), 4449-4453 (2011).</li><li>Balduini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+receptors,+extracellular+proteins+and+myosin+IIA+orchestrate+proplatelet+formation+by+human+megakaryocytes.">Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.</a> Journal of Thrombosis and Haemostasis. 6 (11), 1900-1907 (2008).</li></ol>

Burada, kemik iliğinde yetişen proplateletleri genişletmek için megakaryositlerin verimliliğini değerlendirmek için basit ve düşük maliyetli bir in vitro yöntemi açıklıyoruz. Fare için kemik iliği eksplant modelinin dört ana avantajı vardır. İlk olarak, gelişmiş teknik becerilere gerek yoktur. İkincisi, megakaryosit uzatıcı proplateletler elde etmek için gereken süre oldukça kısadır, eksplant yöntemi için sadece 6 saattir, fare atalarından başlayan geleneksel bir kültür yönte...

Kemik iliği eksplant tekniği ilk olarak 1956 yılında Thiéry ve Bessis tarafından trombosit oluşumunda ilk olay olarak sıçan megakaryosit sitoplazmik uzantılarının oluşumunu tanımlamak için geliştirilmiştir1. Faz kontrastı ve sinematografik teknikleri kullanan bu yazarlar, olgun yuvarlak megakaryositlerin dinamik uzama ve daralma hareketlerini gösteren sitoplazmik uzantılı "kalamar benzeri" trombositojenik hücrelere dönüşümünü karakterize ettiler. Bu kollar, kollar boyunca ve uçlarda küçük şişliklerle filiform olana kadar giderek daha ince hale gelir. İn vitro ve sıvı ortamda elde edilen bu tipik megakaryosit uzamaları, megakaryositlerin sinüzoid duvarlardan kan dolaşımına uzun uzantılar gösterdiği sabit kemik iliğinde gözlenen trombositlerle belirli benzerliklere sahiptir2,3. 1994 yılında TPO'nun keşfi ve klonlaması, kemik iliği eksplantlarında açıklananlara benzeyen proplatelet uzantıları oluşturabilen kültürdeki megakaryositlerin ayırt edilmesine izin verildi4,5,6. Bununla birlikte, megakaryosit olgunlaşması kültür koşullarında çok daha az verimlidir, özellikle kemik iliği olgunlaşmış megakaryositlerin geniş iç zar ağı kültürlü megakaryositlerde az gelişmiştir, trombosit biyogenez mekanizmaları üzerindeki çalışmaları engeller7,8.
Thiéry ve Bessis'e dayanan kemik iliği eksplant modelini, kendi ortamlarında tamamen olgunlaşmış fare megakaryositlerinin gerçek zamanlı proplatlet oluşumunda takip etmek, böylece olası in vitro eserleri ve yanlış yorumlamaları atlatmak için detaylandırırız. Vahşi tip yetişkin farelerde elde edilen sonuçlar, megakaryositlerin proplateletleri genişletme yeteneğini, morfolojilerini ve proplateletlerin karmaşıklığını göstermek için sunulmuştur. Ayrıca megakaryosit kayıt sürecinde veri doğruluğu ve sağlamlığı sağlamak için kalite doğrulaması için hızlı bir niceleme stratejisi sunuyoruz. Burada sunulan protokol, daha önce kitap bölümü olarak yayınlanan yöntemin en son sürümüdür9.

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	<tbody>
		<tr>
			<td>5 mL syringes</td>
			<td>Terumo</td>
			<td>SS+05S1</td>
			<td></td>
		</tr>
		<tr>
			<td>21-gauge needles</td>
			<td>BD Microlance</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2.6H2O</td>
			<td>Sigma</td>
			<td>21108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwall Incubation Chambers</td>
			<td>Electron Microscopy Sciences</td>
			<td>70324-02</td>
			<td>Depth : 0,2 mm</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H-3375</td>
			<td>pH adjusted to 7.5</td>
		</tr>
		<tr>
			<td>Human serum albumin</td>
			<td>VIALEBEX</td>
			<td>authorized medication : n&#176; 3400956446995</td>
			<td>20% (200mg/mL -100mL)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma</td>
			<td>BVBW8448</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72200-40</td>
			<td>22 mm x 55 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica Microsystems SA, Westlar, Germany</td>
			<td>DMI8 - 514341</td>
			<td>air lens</td>
		</tr>
		<tr>
			<td>microscope camera</td>
			<td>Leica Microsystems SA, Westlar, Germany</td>
			<td>K5 CMS GmbH -14401137</td>
			<td>image resolution : 4.2 megapixel</td>
		</tr>
		<tr>
			<td>Mouse serum</td>
			<td>BioWest</td>
			<td>S2160-010</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>PSG 100x</td>
			<td>Gibco, Life Technologies</td>
			<td>1037-016</td>
			<td>10,000 units/mL penicillin, 10,000 &#956;g/mL streptomycin and 29.2 mg/mL glutamine</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose D (+)</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
	</tbody>
</table>

proplatelet formation dynamics of mouse fresh bone marrow explants

Megakaryopoezisin son aşaması, proplateletler olarak adlandırılan olgun megakaryositlerden sitoplazmik uzantılara yol açar. In vitro-farklılaştırılmışmegakaryositler kullanılarak proplatlet oluşumu hakkında çok şey öğrenilmiştir; bununla birlikte, geleneksel kültür sistemlerinin kemik iliğinin içinde gerçekleşen farklılaşma/olgunlaşma sürecini sadık bir şekilde geri almadığına dair artan bir kanıt vardır. Bu yazıda, thiéry ve Bessis tarafından 1956 yılında, kendi çevrelerinde olgunlaşmış megakaryositleri görselleştirmek, böylece potansiyel eserleri ve yanlış yorumlamaları atlatmak için açık bir yöntem sunuyoruz. Taze kemik ilikleri, farelerin uyluklarının yıkanmasıyla toplanır, 0,5 mm kesitler halinde dilimlenir ve fizyolojik tampon içeren 37 °C'de bir kuluçka odasına yerleştirilir. Megakaryositler eksiz çevrede yavaş yavaş görünür hale gelir ve bir video kameraya bağlı ters bir mikroskop altında 6 saate kadar gözlenir. Zamanla, megakaryositler şekillerini değiştirir, bazı hücreler küresel bir forma sahip ve diğerleri kalın uzantılar geliştirir veya birçok ince proplatlet geniş dallanma ile genişletir. Hem nitel hem de nicel araştırmalar yapılmaktadır. Bu yöntem, çok sayıda megakaryosit mevcut olduğu için basit, tekrarlanabilir ve hızlı olma avantajına sahiptir ve klasik olarak yarısı, kültürlü fare megakaryositleri için 4 güne kıyasla 6 saatte proplatelet oluşturur. Mutant farelerin çalışmasına ek olarak, bu yöntemin ilginç bir uygulaması, farmakolojik ajanların kültürlerde oluşabilecek farklılaşma sürecine müdahale etmeden proplatelet uzatma işlemi üzerinde basit bir şekilde değerlendirilmesidir.

Nitel sonuçlar. Deneyin başında tüm hücreler kemik iliği bölümünde sıkıştırılıyor. Hücrelerin eksplantların çevresinde net bir şekilde görünür hale gelmesi 30 dakika sürer. Megakaryositler daha sonra büyük boyutlarıyla tanınabilir ve evrimleri zamanla incelenebilir (boyut, şekil, dinamik, proplatlet uzantısı ve trombosit salınımı) (Şekil 2A). Küçük megakaryositlerin çapı 20 ila 30 μm arasındadır ve çekirdekleri polilobulated iken ol...

Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

Burada, fizyolojik ortamlarında farklılaşmış megakaryositlerin proplatlet oluşturma yeteneğini değerlendirmek için numune hazırlamadan mikroskobik slayt analizine kadar kemik iliği eksplant yöntemini detaylandırıyoruz.

Fare Taze Kemik İliği Eksplantlarının Proplatelet Formasyon Dinamikleri

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

Bu prosedürün genel amacı, kendi ortamlarında olgunlaşan fare megakaryositlerinden proplatelet oluşumunu gerçek zamanlı olarak takip etmektir. Bu yöntem basit, tekrarlanabilir ve hızlı olma avantajına sahiptir. Birçok megakaryosit analiz edebilir ve profilaklet oluşumunu sadece altı saat içinde görselleştirebilir, bunun yerine kültürün ilk gününü beklemek yerine fare megakaryositleri in vitro.Bu yöntemin ilginç bir uygulaması, özellikle proplatelet uzantısı adımında farmasötik tedavinin veya genetik mutasyonun etkisini incelemek için bir olasılıktır. Burada in-vitro kültürde olduğu gibi farklılaşma sürecine müdahale yoktur. Bu video, kemik iliğini yıkamanın ve kesmenin bu önemli adımlarını sağlamaya yardımcı olur.Ayrıca, trombofilik hastalıkta kusurların karakterizasyonu için gerçekten yararlı olan morfoloji ve megakaryositlerin nasıl sınıflandırılacağı açıklanmıştır. Başlamak için, Tyrode'un tamponu 37 santigrat derecede ısıtın ve sıcaklığı 37 santigrat dereceye getirmek için mikroskobun ısıtma odasını açın. Zamanlayıcı, kuluçka odaları, beş mililitre 21 kalibre şırıng, forseps, jilet, Pasteur pipetler, cam kaydıraklar ve 15 mililitre santrifüj tüpü gibi gerekli tüm araçları hazırlayın.Diz tarafındaki uyluk kemiğinin açıklığı içine 21 kalibrelik bir iğne sokarak kemik iliğini iki mililitre Tyrode tamponu ile yıkayın ve sağlam bir ilik silindirini almak için pistona yavaşça bastırın. Sağlam kemik iliğini dikkatlice ve nazikçe cam bir kaydırağa aktarmak için üç dakikalık litrelik plastik pipet kullanın. Yıkama anında sıkıştırılmış olabilecek iliğin uçlarını kesin.Daha sonra megakaryositlere zarar vermemek için keskin bir jilet kullanarak 0,5 milimetre kalınlığında ince transversal bölümleri kesin. Plastik bir pipet kullanarak, Tyrode'un tamponu içeren bir mililitrelik bir tüpe 10 bölüm toplayın. Bölümleri dikkatlice 13 milimetre çapında bir kuluçka odasına aktarın.Tamponu epire edin ve hacmi%5 fare serumu ile desteklenmiş 30 mikrolitre Tyrode tamponu olarak ayarlayın. Bölümleri bir mesafede konumlandırın. Hava kabarcıklarının oluşumunu önlemek için kapak kaymasını eğimli olarak 22 ila 55 milimetrelik bir kapak kayması ile kendinden yapışkanlı odayı kapatın.Odayı ısıtma odasına 37 santigrat derecede yerleştirin. Kronometreyi başlatın ve deneyi 37 santigrat derecede altı saat çalıştırın, Megakaryositlerin dönüşümini kaydetmek için videolar yapın. 30 dakika sonra, ilik hücreleri yavaş yavaş bir mono tabaka oluşturan eksplantın çevresine göç etti.Bir saatlik inkübasyondan sonra, megakaryositler büyük boyutları ve polilobulated çekirdekleri ile tanımlanabilir. Megakaryositlerin sayısı üç saatlik inkübasyondan sonra artar ve bazılarının uzun uzantıları vardır. Kuluçka odasındaki her bölümü yerelleştirmek için bir harita çizin.Bir saat sonra, her bölümün çevresindeki dev polilobulated hücreler olan görünür megakaryositleri tanımlayın ve çizimdeki konumlarını çizin. Üç ve altı saat sonra bu işlemi tekrarlayın. Megakaryositler elle sayılır ve inkübasyon odasının sızdırmazlığından üç ve altı saat sonra morfolojilerine göre sınıflandırılır.Küçük, kalın niyetli büyük ve proplatelet uzatma olarak sınıflandırılırlar. Haritalama yardımıyla, evrimleri zaman içinde takip edilebilir. Sonuçlar, her gözlem zamanında her sınıfın yüzdesi olarak ifade edilir.Klasik olarak, çevrede görülebilen megakaryositlerin yarısı, vahşi tip fare kemik iliği için proplateletleri altı saatte uzatır. Yuvarlak megakaryositlerin kaderi, zaman içinde proplateletleri nasıl oluşturduklarını görmek için sıralı görüntüler yakalayarak takip edildi. Bu prosedürü denerken hatırlanması gereken önemli bir şey, eksplantları deney süresince 37 santigrat derecede tutmaktır.Farklı bir sıcaklık sonuçları değiştirebilir. İlginçtir ki, explant protokolü içe dönük mikroskopi deneylerinden sonra kullanılabilir. Sonunda kemik iliği eksplantlarındaki megakaryositler doğal olarak floresan olacak ve davranışları kolayca araştırılabilir.Bu, aynı hayvanlar üzerinde farklı tedaviyi birleştirmeyi ve böylece fare sayısını azaltmayı mümkün khaline getirir. Sonuç olarak, explant yöntemi hızlı, basittir ve doğal megakaryositlerin dış proplateletlere kapasitesi hakkında birçok bilgi sağlar. Ortaya çıkan nitel ve nicel bulgu trombofilik hastalığı anlamamızın anahtarıdır.fizyolojik veya patolojik bağlamda.

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Biology

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 ...

Biyoloji

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

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

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Kemik İliği Hematopoetik Hücreler, Homing

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

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

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Fare Kemik İliği osteoklast türetilmesi

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

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

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Sıçan kemik Iliği Osteoclast precursors yalıtım, arıtma ve farklılaşma

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

In Vivo Fare Kafatası Kemik İliği'nde Megakaryositlerin ve Proplateletlerin İki FotonLu Görüntülemesi

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...