Name: in vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy
Uploaded: 2014-08-06T16:00:00
Description: Watch this Scientific Journal Video about In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy at JoVE.com

This work was supported by the Intramural Research Program of the National Heart, Lung, Blood Institute of the National Institutes of Health. We thank Boris Fehse (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for providing the five LeGO vector plasmids; Christian A. Combs and Neal S. Young (NHLBI, NIH) for discussions, support and encouragement throughout this study, and Andre LaRochelle (NHLBI, NIH) for assistance with tail vein injections.

Tüm fareler ev sahipliği ve Bakım ve Sağlık Ulusal Enstitüleri Laboratuvar Hayvanları Kullanım Kılavuzu uyarınca ele, ve bir UKAKE Hayvan Bakım ve Kullanım Kurulu onaylı protokol alındı. Erkek B6.SJL-Ptprc (d) Pep3, (b) / 6-12 haftalık BoyJ (B6.SJL) ve C57BL / 6 fareler, sırasıyla, verici ve alıcı olarak kullanılmıştır. Malzemeler, reaktifler ve ekipmanın bir listesi Tablo 1&#39;de verilmektedir. Fare HSPCs 1. Lego İletimi...

<ol>
	<li>Lo Celso, ., C, D. T., Scadden, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+haematopoietic+stem+cell+niche+at+a+glance.">The haematopoietic stem cell niche at a glance.</a> J Cell Sci. 124, 3529-3535 (2011).</li><li>Lo Celso, 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=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457, 92-96 (2009).</li><li>Takaku, 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=Hematopoiesis+in+3+dimensions+human+and+murine+bone+marrow+architecture+visualized+by+confocal+microscopy.">Hematopoiesis in 3 dimensions human and murine bone marrow architecture visualized by confocal microscopy.</a> Blood. 116, e41-e55 (2010).</li><li>Malide, D., Metais, J. Y., Dunbar, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clonal+analysis+of+murine+hematopoietic+stem+and+progenitor+cells+marked+by+5+fluorescent+proteins+using+confocal+and+multiphoton+microscopy.">Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy.</a> Blood. 120, e105-e116 (2012).</li><li>Weber, K., Mock, U., Petrowitz, B., Bartsch, U., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+gene+ontology+(LeGO)+vectors+equipped+with+novel+drug-selectable+fluorescent+proteins+new+building+blocks+for+cell+marking+and+multi-gene+analysis.">Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins new building blocks for cell marking and multi-gene analysis.</a> Gene Ther. 17, 511-520 (2010).</li><li>Weber, K., Bartsch, U., Stocking, C., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicolor+panel+of+novel+lentiviral+'gene+ontology'+(LeGO)+vectors+for+functional+gene+analysis.">A multicolor panel of novel lentiviral 'gene ontology' (LeGO) vectors for functional gene analysis.</a> Mol Ther. 16, 698-706 (2008).</li><li>Weber, 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=RGB+marking+facilitates+multicolor+clonal+cell+tracking.">RGB marking facilitates multicolor clonal cell tracking.</a> Nat Med. 17, 504-509 (2011).</li><li>Lo Celso, ., Lin, C. P., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+transplanted+hematopoietic+stem+and+progenitor+cells+in+mouse+calvarium+bone+marrow.">In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow.</a> Nature protocols. 6, 1-14 (2011).</li><li>Kohler, 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=Altered+cellular+dynamics+and+endosteal+location+of+aged+early+hematopoietic+progenitor+cells+revealed+by+time-lapse+intravital+imaging+in+long+bones.">Altered cellular dynamics and endosteal location of aged early hematopoietic progenitor cells revealed by time-lapse intravital imaging in long bones.</a> Blood. 114, 290-298 (2009).</li><li>Campagnola, P. J., 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=Three-dimensional+high-resolution+second-harmonic+generation+imaging+of+endogenous+structural+proteins+in+biological+tissues.">Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.</a> Biophys J. 82, 493-508 (2002).</li><li>Weigert, R., Sramkova, M., Parente, L., Amornphimoltham, P., Masedunskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+a+novel+tool+to+study+cell+biology+in+living+animals.">Intravital microscopy a novel tool to study cell biology in living animals.</a> Histochem Cell Biol. 133, 481-491 (2010).</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, 238-250 (2012).</li></ol>

Biz canlı dokularda hacimsel ve dinamik görüntüleme elde etmek için tandem confocal ve 2-foton mikroskopi ile 5FP kodlayan LEGO vektörler ve görüntüleme ile işaretleme kombinasyonal tarafından oluşturulan büyük çeşitliliğini birleştirerek, burada son zamanlarda klonal hücre izleme için tasarlanmış güçlü bir metodoloji ayrıntılarını anlatacağız. Bu 5 &quot;farklı&quot; 8-bit kanallarda confocal spektral kimliğini kaydederek işaretleme FP-tabanlı renk kullanımı genişletilmiş. Böylec...

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.
This was corrected in the discussion from:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 30 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.
to:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 300 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.

Erratum: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Kan hücreleri olarak adlandırılan hematopoiezin üretimi, hematopoietik kök ve projenitör hücreleri (HSPCs) küçük bir nüfus tarafından muhafaza edilmektedir. Bu hücreler tüm kendini yenileme ve farklılaşma 1,2 kontrolünde rol, osteoblastlar, stromal hücreler, yağ dokusu ve vasküler yapılardan oluşan bir kompleks microenvironmental niş içinde kemik iliği (BM) içinde bulunur. Sağlam BM doğrudan gözlemlere geleneksel erişilemez olmuştur gibi, HSPC ve mikroçevresinin arasındaki etkileşimler in vivo ölçüde uncharacterized kalır. Daha önce, konfokal floresan ve yansıma mikroskobu 3 kullanılarak sağlam BM&#39;nin 3D mimarisi görselleştirmek için bir metodoloji kurdu. Biz BM EGFP-işaretli HSPCs genişlemesi, özelliği, ancak yalnızca tek bir FP kullanımı klonal düzeyde rejenerasyonu tespiti engellemiştir. Çok yakın zamanda biz kurucu fl ifade Lentiviral Gen Ontoloji (Lego) yararlandı vektörleri aldıuorescent proteinler (FP) hücreleri verimli 4,5 işaretlemek için. 3 ya da 5 vektörlerle HSPC Co-transdüksiyon çoklu bireysel klonların HSPC izleme sağlayan, birleşik bir renk paleti çeşitli oluşturur. İşaretli HSPC ışınlanmış farelerde BM bireysel HSPC homing ve bütünleşmesini izlemek için izin yeni görüntüleme teknolojisi ile birlikte. Lego vektörler kemik ve diğer açıkça görüntülenmesini sağlayan, (Cerulean, eGFP, Venüs, tdTomato ve mCherry itibaren) 3 veya 5 farklı aile hekimleri ile HSPC işaretlemek ve konfokal ve 2-foton mikroskopi BM zamanla kendi naklini takip etmek kullanıldı matris yapıları ve bunların mikro bileşenlerine HSPC klonların ilişkisi. HSPC lego vektörleri ile transduse edilmiş C57BL / 6 fareleri BM den soy negatif hücreleri gibi arıtıldı ve miyelo ablasyon konjenik farelerine kuyruk damarından enjeksiyon ile yeniden infüze edildi. Sternum BM doku büyük hacimli izleyerek biz erken meydana endesiteal bütünleşmesini görüntülendiBM rejenerasyon. Farklı renkli spektrumları ile hücre kümeleri kemiğe yakın ilk olarak ortaya çıktı ve zaman içinde merkezi olarak ilerledi. İlginç bir şekilde, en fazla 3 hafta sonra kemik iliği, kan akışı yoluyla oldukça yaygın HSPCs yayılması daha yakın olarak kemik iliğinde türetilen bireysel HSPCs hematopoez yayılmasını gösteren makroskopik klonal kümeleri oluşturmuştur. Daha az renk çeşitliliği çok vektörler ile hücrelerin repopulating uzun vadeli transduse güçlük göstermektedir de, geç zaman noktalarında oldu. Buna ek olarak, HSPCs, dalakta farelerde hematopoez de sorumlu bir organ kümeleri oluşturmuştur. HPSC türetilmiş ayrı ayrı hücreler yanı sıra, timus, lenf düğümleri, dalak, karaciğer, akciğer, kalp, cilt, iskelet kas, yağ dokusu ve böbrek çözülebilirdi. 3D görüntüler dokuların en az değişimleri ile hücrelerinin dağılımını takdir nicel ve nitel olarak değerlendirilebilir. Son olarak, tasvirrezonans tarama multiphoton ve konfokal time-lapse görüntüleme birleştirerek 4D canlı dinamik çalışmaların d fizibilite. Bu metodoloji doku rejenerasyonu ve patoloji çalışmada güçlü bir araç sağlayarak, onların bozulmamış 3D mimarisi Işıksal işaretlenmiş hücreler nüfus non-invaziv, yüksek çözünürlüklü, çok boyutlu hücre kaderini izlemeyi sağlar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Production of viruses</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-Cer2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27338</td>
			<td>Expression of Cerulean</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-G2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">25917</td>
			<td>Expression of eGFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-V2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27340</td>
			<td>Expression of Venus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-T2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27342</td>
			<td>Expression of tdTomato</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-C2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27339</td>
			<td>Expression of mCherry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Calciumm Phosphate Transfection Kit</td>
			<td>Sigma</td>
			<td>CAPHOS-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Tissue culture dish</td>
			<td style="width:493px;">BD Falcon</td>
			<td style="width:216px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">IMDM</td>
			<td style="width:493px;">Gibco, Life Technology</td>
			<td style="width:216px;">12440-053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Fetal Bovine Serum Heat Inactivated</td>
			<td style="width:493px;">Sigma</td>
			<td style="width:216px;">F4135-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pen Strep Glutamine</td>
			<td style="width:493px;">Gibco, Life Technology</td>
			<td style="width:216px;">10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Centrifuge Tubes</td>
			<td style="width:493px;">Beckman Coulter</td>
			<td style="width:216px;">326823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">SW28 Ultracentrifuge</td>
			<td style="width:493px;">Beckman Coulter</td>
			<td style="width:216px;">L60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Millex Syringe Driven Filter Unite (0.22um)</td>
			<td style="width:493px;">Millipore</td>
			<td style="width:216px;">SLGS033SS</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse cell collection, purification, transduction and transplantation</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">ACK lysing buffer</td>
			<td style="width:493px;">Quality Biologicals Inc.</td>
			<td style="width:216px;">118-156-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Lineage Cell Depletion Kit (mouse)</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-090-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LS columns + tubes</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-041-306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pre-Separation Filters (30um)</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:493px;">StemSpan SFEM serum-free medium for culture and expansion of hematopoietic cells (500mL)</td>
			<td>StemCell Technologies Inc&#160;</td>
			<td>9650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">murine IL-11 CF</td>
			<td>R &#38; D Systems Inc</td>
			<td>418-ML-025/CF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">recombinant murine SCF</td>
			<td style="width:493px;">RDI Division of Fitzgerald Industries Intl&#160;</td>
			<td>RDI-2503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">recombinant murine IL-3</td>
			<td>R &#38; D Systems Inc</td>
			<td>403-ML-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">FLT-3 Ligand</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-096-480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">12-well plates, Costar</td>
			<td style="width:493px;">Corning Inc.</td>
			<td style="width:216px;">3527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Retronectin</td>
			<td style="width:493px;">Takara Bio Inc</td>
			<td style="width:216px;">T100A/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Protamine Sulfate</td>
			<td style="width:493px;">Sigma</td>
			<td style="width:216px;">P-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Confocal and two-photon microscopy</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">DMEM</td>
			<td style="width:493px;">Lonza</td>
			<td style="width:216px;">12-614F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">1M Hepes</td>
			<td style="width:493px;">Cellgro, Mediatech Inc.</td>
			<td style="width:216px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Glass bottom culture dish P35G-0-20-C</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Glass bottom culture dish P35G-0-14-C</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Nunc Lab-Tek Chambered Coverglass #1 Borosilicate coverglass; 4-well</td>
			<td>Thermo Scientific</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Leica TCS SP5 AOBS five channels confocal and multi-photon microscope</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chameleon Vision II -TiSaph laser range 680-1080nm</td>
			<td style="width:493px;">Coherent</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chameleon Compact OPO laser range 1030-1350nm</td>
			<td style="width:493px;">Coherent</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCX-IRAPO-L 25x/0.95 NA water dipping objective (WD=2.5 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HC-PLAPO-CS 20x/0.70 NA dry objective (WD=0.6 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HC-PL-IRAPO 40x/1.1NA water immersion objective (WD=0.6 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Imaris software version 7.6</td>
			<td style="width:493px;">Bitplane</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy

Biz geliştirdik ve fare kemik iliği nakli deneysel model kullanılarak in vivo hematopozun incelemek için yüksek uzaysal ve zamansal çözünürlüğe sahip hematopoetik kök hücrelerin (HSPCs) klonal takibi için metodoloji işaretleme floresan valide. Floresan proteinleri (FP) kodlayan Lentiviral vektörleri kullanılarak işaretleme Genetik kombinatoryel ileri mikroskopi görüntüleme yoluyla hücre kaderi haritalama etkin. Beş farklı FPS kodlayan vektörler: Shibuya, EGFP Venüs tdTomato ve MCherry renk işaretlenmiş hücrelerin çeşitli bir palet oluşturma, aynı zamanda HSPCs transdüksiyonu için kullanılmıştır. Konfokal / iki foton hibrid mikroskopi kullanılarak Görüntüleme aynı anda yüksek çözünürlüklü benzersiz işaretli hücrelerin değerlendirilmesi ve dokuların yapısal unsurları ile birleşik olarak da döl sağlar. Hacimsel spektral kodlu HSPC türetilmiş hücreler invasiv olmayan bir kemik iliği dahil olmak üzere çeşitli dokulara, tespit edilebilir olduğunu ortaya koymaktadır (büyük alanlar üzerinde analiz BM), Transplantasyon aşağıdaki kapsamlı bir süre için. 4D video hızı multiphoton ve konfokal time-lapse görüntüleme birleştirerek Canlı çalışmalar nicel bir şekilde dinamik hücresel ve klonal izleme olasılığını göstermektedir.

Tüm montaj 3D konfokal sternum kemik iliği zaman tabii / 2-foton mikroskopi rekonstrüksiyon olağanüstü özelliklere sahip bir desen uyumunu ve nakledilen ko-5fps&#39;dir genişlemesini ortaya: klon açıkça belirlenen, homojen başlangıçta geniş renk paleti ve ilerleme ile işaretlenmiş çıktı zamanla tercihen çoğunlukla tek renk hücreleri içeren. Konfokal mikroskopi kurulum ve sternal kemik iliği görüntüleme 5fps&#39;dir-işaretli HSPC temsili örnekleri, Şekil 2 ve Film 1...

Watch this Scientific Journal Video about In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy at JoVE.com

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Hematopoetik kök hücrelerin işaretleme Kombinatoryel 5 floresan proteinleri rejenerasyon sırasında kemik iliği hematopoetik mimarisi içine anlayışlar, confocal ve iki foton mikroskopi ile in vivo klonal izleme sağlar. Bu yöntem nakli aşağıdaki kapsamlı bir süre için sağlam dokularda spektral kodlu HSPCs kökenli hücrelerin non-invaziv kader haritalama sağlar.

Eşodaklı ve multiphoton Mikroskopi kullanarak beş Floresan Proteinleri ile işaretlenmiş Hematopoetik kök hücrelerin in vivo klonal Takip

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Research

JoVE Journal

Biology

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

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

Biyoloji

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Küçük Hayvan Kas Aşı dinamiği ile Takip In Vivo Floresan Görüntüleme

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

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 ...

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

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

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

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

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

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) ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Ex-Vivo Konfokal Mikroskopi ile Lenf Nodal Yapısı nın Ve Hücresel Lokalizasyonun Görselleştirilmesi

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

Efflux pompaları Inhibisyonu Ile hematopoetik kök ve progenitor hücrelerde mitokondriyal membran potansiyelinin akış Cytometrik değerlendirilmesi doğruluğu iyileştirilmesi

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

Hematopoetik Kök Hücrelerde ve T-Hücrelerinde Mitokondriyal Kitle ve Membran Potansiyelinin Akış Sitometrisi ile Ölçülmesi

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Hücre Dışı Akı Analizörü Kullanılarak Fare Hematopoetik Kök ve İlkel Progenitör Hücrelerinde Hücresel Biyoenerjetik Verilerin Değerlendirilmesi

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...