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

저자들은 장 이브 린켈, 줄리 보셔, 패트리샤 래퍼, 모니크 프룬드, 케티 크네즈-히퍼트에게 기술 지원을 부탁드립니다. 이 작품은 ANR (기관 국가 드 라 Recherche) 그랜트 ANR-17-CE14-0001-01 및 ANR-18-CE14-0037에 의해 지원되었습니다.

모든 동물 실험은 유럽 표준 2010/63/EU및 스트라스부르 대학의 동물 실험 윤리에 대한 CREMEAS 위원회 (코미테 레지오날 데티크 앙 마티에르 디에리포앙 마티에르 데테리션 애니멀 스트라스부르)에 따라 수행되었습니다.
1. 시약 준비
<ol><li>표 1에설명된 대로 시약을 준비한다.<ol>
<li>스톡 I의 경우 각 분말을 별도로 녹입니다. 준비의 진동이 295 mOsm / L보다 높은지 확?...

<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>

여기에서 우리는 골수에서 성장한 프로혈소를 확장하기 위하여 거대 karyocytes의 효율성을 평가하는 간단하고 저렴한 체외 방법을 기술합니다. 마우스에 대한 골수 각질 모델은 네 가지 주요 장점이 있습니다. 첫째, 고급 기술 기술이 필요하지 않습니다. 둘째, 메가카요세포-연장 프로플라틀을 얻는 데 필요한 시간은 매우 짧고, 절제 방법에 대해서만 6시간밖에 되지 않는데, 이는 마우스 전?...

골수 각질 기술은 1956년 티에리와 베시스에 의해 처음 개발되어 혈소판 형성1의초기 이벤트로서 쥐 메가카요세포 세포질 확장의 형성을 기술하였다. 위상 대비 및 촬영 기술을 사용하여, 이 저자는 신장과 수축의 동적 움직임을 보여주는 세포질 확장과 "오징어 같은"혈전 세포로 성숙한 둥근 메가 카요세포의 변환을 특징으로. 이 팔은 팔을 따라 그리고 끝에서 작은 붓기와 filiform될 때까지 점진적으로 얇아집니다. 시험관 및 액체 매체에서 얻어진 이러한 전형적인 메가카요세포 신장은 고정형 골수에서 관찰된 혈소판과 일정한 유사성을 가지며, 여기서 거대 카르요퀴트는 혈중 벽으로 시누치성 벽을 통해 긴 연장을 돌출하여 혈액 순환2,3로돌출된다. 1994년 TPO의 발견 및 복제는 골수 에 기재된 것과 유사한 프로플라틀 확장을 형성할 수 있는 배양에서 메가카르요사이클을 구별할 수 있게4,5,6. 그러나, 메가카요세포 성숙은 배양 조건에서 훨씬 덜 효율적이며, 특히 골수 성숙 메가카요세포의 광범위한 내부 멤브레인 네트워크는 배양 된 메가 카요사이클로 저개발되어 혈소판 생물 발생7,8의메커니즘에 대한 연구를 방해한다.
우리는 여기에 Thiéry와 Bessis에 따라 골수 박멸 모델을 자세히 설명, 마우스 메가 카요 사이클의 실시간 proplatelet 형성에 따라, 이는 완전히 자신의 네이티브 환경에서 성숙한, 따라서 체외 유물과 오해에서 가능한 우회. 야생 형 성인 마우스에서 얻은 결과는 프로 플라틀릿, 그들의 형태 및 프로 플라츠의 복잡성을 확장하는 메가 카요사이클의 능력을 설명하기 위해 제시된다. 또한 메가카요세포 기록 과정에서 데이터 정확성과 견고성을 보장하기 위해 품질 검증을 위한 신속한 정량화 전략을 도입했습니다. 여기에 제시된 프로토콜은 이전에 책 장9로출판된 방법의 최신 버전입니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

메가카요포이시스의 마지막 단계는 성숙한 거대 카르요세포, 소위 프로플라츠에서 세포질 확장으로 이어집니다. 체외에서 -분화 된 메가 카요세포를 사용하여 프로 플라츠판 형성에대해 많은 것을 배웠습니다. 그러나, 기존의 문화 시스템이 골수 내부의 장소를 취하는 분화 / 성숙 과정을 충실하게 재구성하지 않는다는 증거가 증가하고 있다. 이 원고에서는 1956년 티에리와 베시스가 원래 설명한 절묘한 방법을 제시하여 자신의 토착 환경에서 성숙한 거대 카르요세포들을 시각화하여 잠재적인 유물과 오해를 우회합니다. 신선한 골새끼는 마우스의 대퇴골을 플러시하여 수집되고, 0.5mm 단면으로 슬라이스하고, 생리적 완충액을 함유하는 37°C의 인큐베이션 챔버에 배치된다. 메가카요세포는 축출 주변에서 점진적으로 볼 수 있게 되고 비디오 카메라에 결합된 반전된 현미경으로 최대 6시간까지 관찰됩니다. 시간이 지남에 따라, 메가 카요 세포는 구형 형태를 가지고 다른 두꺼운 확장을 개발하거나 광범위한 분기와 많은 얇은 proplatelet을 확장, 모양을 변경합니다. 정성적 및 정량적 조사가 모두 수행됩니다. 이 방법은 수많은 거대 카르요세포가 존재하므로 간단하고 재현 가능하며 빠르며, 그 중 절반은 배양 마우스 메가카요세포에 대한 4일에 비해 6시간 만에 프로플라틀렛을 형성한다는 장점이 있다. 돌연변이 마우스의 연구 외에도, 이 방법의 흥미로운 적용은 배양에서 발생할 수 있는 분화 과정을 방해하지 않고, 프로플라틀 확장 과정에 대한 약리학제제의 간단한 평가이다.

질적 결과. 실험의 시작 부분에서, 모든 세포는 골수 단면도에서 압축됩니다. 세포가 각성의 주변에서 명확하게 보이려면 30분이 걸립니다. 메가카요세포는 큰 크기로 인식될 수 있으며, 그 진화는 시간이 지남에 따라 연구될 수 있다(크기, 모양, 동적, 프로혈소판 연장 및 혈소판 방출)(그림2A). 작은 거대 카르요세포는 직경 20에서 30 μm 사이이며, 성숙한 둥근 메?...

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

여기서, 우리는 골수 각질 방법을 자세히, 샘플 준비에서 현미경 슬라이드 분석에, 프로 플라츠판을 형성하는 그들의 생리 환경에서 분화 한 메가 카요 세포의 능력을 평가하기 위해.

마우스 신선한 골수 이별의 프로플라츠 형성 역학

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

이 절차의 전반적인 목적은 그들의 네이티브 환경에서 성숙한 마우스 거대 카르요세포에서 proplatelet의 형성을 실시간으로 따르는 것입니다. 이 방법은 간단하고 재현 가능하며 빠르다는 장점이 있습니다. 하나는 많은 메가 카요 사이클을 분석하고 시험관내 마우스 거대 카르요세포에 관해서는 문화의 첫날을 기다리는 대신 6 시간 만에 프로 플라플라인의 형성을 시각화 할 수 있습니다.이 방법의 흥미로운 응용 프로그램은 특히 프로 플라틀렛 확장의 단계에서 약제 학적 치료 또는 유전 돌연변이의 효과를 연구 할 수있는 가능성입니다. 여기서는 체외 문화의 경우와 같이 분화 과정에 간섭이 없습니다. 이 비디오는 골수를 플러싱하고 절단하는 이러한 주요 단계를 보장하는 데 도움이됩니다.또한 혈전성 질환의 결함 특성화에 정말 유용한 형태와 거대 카르요사이클을 분류하는 방법에 대해서도 설명했습니다. 우선, Tyrode의 버퍼를 섭씨 37도에서 따뜻하게 하고 현미경의 난방 실을 켜서 온도를 섭씨 37도까지 끌어올립니다. 타이머, 인큐베이션 챔버, 5 밀리리터 21 게이지 주사기, 집게, 면도날, 파스퇴르 파이펫, 유리 슬라이드 및 15 밀리리터 원심분리기 튜브와 같은 필요한 모든 도구를 준비합니다.21 게이지 바늘을 무릎 쪽의 대퇴골 개구부에 도입하여 티로드의 완충제 두 밀리리터로 골수를 씻어내고 플런저를 천천히 눌러 그대로 골수 실린더를 회수합니다. 3분 짜리 플라스틱 파이펫을 사용하여 그대로 있는 골수를 유리 슬라이드에 조심스럽게 부드럽게 전달합니다. 플러시 에 압축 되었을 수 있습니다 골수의 끝을 잘라.그런 다음 날카로운 면도날을 사용하여 0.5mm 두께의 얇은 대지 부분을 잘라 메가 카요사이클을 손상시키지 않도록합니다. 플라스틱 파이펫을 사용하여 티로드의 버퍼가 들어 있는 1밀리리터 튜브에 10개의 섹션을 수집합니다. 조심스럽게 13 밀리미터의 직경을 가진 잠복챔버로 섹션을 전송합니다.버퍼를 흡인하고 5%의 마우스 세럼으로 보완된 Tyrode의 버퍼30 마이크로리터로 볼륨을 조정합니다. 단면을 멀리 배치합니다. 22~ 55mm 커버 슬립으로 자체 접착제 챔버를 밀봉하여 공기 포기의 형성을 피하기 위해 커버 슬립을 절제합니다.챔버를 섭씨 37도에 가열실에 놓습니다. 크로노미터를 시작하고 섭씨 37도에서 6시간 동안 실험을 실행하여 메가카르요세포의 변형을 기록하기 위해 비디오를 만듭니다. 30분 후, 골수 세포는 모노층을 형성하는 해종의 주변으로 점차 이동한다.1시간 동안 배양한 후, 메가카요세포는 큰 크기와 폴리로부트된 핵으로 식별할 수 있다. 메가카요세포의 수는 3시간 동안 인큐베이션을 한 후 증가하고 일부는 긴 연장을 가지고 있습니다. 맵을 그려 인큐베이션 챔버의 각 섹션을 지역화합니다.1시간 후 각 섹션 의 주변에 거대한 폴리로직화된 세포인 눈에 보이는 메가카요세포를 식별하고 도면에 위치를 그립니다. 3시간 6시간 후에 이 절차를 반복합니다. 메가카요세포는 인큐베이션 챔버를 밀봉한 후 3시간 6시간 후에 그들의 형태에 따라 수동으로 계산되고 분류된다.그들은 작은 으로 분류 됩니다., 두꺼운 의도, 그리고 프로 플래틀렛 확장. 매핑의 도움으로 시간이 지남에 따라 진화를 따를 수 있습니다. 결과는 각 관측 시간에 각 클래스의 백분율로 표시됩니다.고전적으로, 주변에서 보이는 거대 카르요세포의 절반은 야생형 마우스 골수의 경우 6시간 동안 프로플라츠를 확장한다. 둥근 메가카요세포의 운명은 시간이 지남에 따라 순차적인 이미지를 캡처하여 프로플라츠판을 형성하는 방법을 이미지했습니다. 이 절차를 시도 할 때 기억해야 할 중요한 것은 실험 기간 동안 섭씨 37도에서 이절을 유지하는 것입니다.온도가 다른 온도로 결과를 변경할 수 있습니다. 흥미롭게도, 외래 프로토콜은 내향성 현미경 실험 후에 사용될 수 있습니다. 결국 골수 의 거대 카르요세포는 자연적으로 형광이 될 것이며, 그들의 행동은 쉽게 조사 될 수 있습니다.이것은 가능하게 동일 동물에 다른 처리를 결합하고 마우스의 수를 감소시다. 결론적으로, 이 절제 방법은 빠르고 간단하며 외부 프로플라틀에 천연 거대 핵세포의 용량에 대한 많은 정보를 제공한다. 결과 질적 및 정량적 발견은 혈전성 질병의 우리의 이해에 열쇠입니다.생리학적 또는 병리학적 맥락에서.

proplatelet-formation-dynamics-of-mouse-fresh-bone-marrow-explants

Research

JoVE Journal

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.

골수 엽 성의 전구 물질에서 골수양 돌기 세포의 문화

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

연구

생물학

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.

골수의 생성은 2 광자 영상에서 사용하기 위해 Murine 돌기 세포를 유래

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.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

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.

골수에 조혈 세포의 유도

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.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

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.

마우스 골수에서 파골 세포의 유도

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.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

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.

Murine 골 수 유래 수지상 세포와 대 식 세포에 형광 성 융해 단백질의 표정

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.

쥐 골 골에서의 파 골 세포 전 구체의 분리, 정제 및 분화

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.

인 비보 마우스 두개골 골수에 있는 메가카르요세포와 프로플라틀의 2광화상

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