Forfatterne takker Research Flowcytometri Core på Cincinnati Children Hospital Research Foundation og Richard Demarco, Sherree Ven, og Scott Mordokaj fra Amnis Corporation (en del af EMD Milllipore) for sagkyndig teknisk support. Dette arbejde blev støttet af National Institutes of Health tilskud K08HL088126 og R01HL116352 (TAK) og P30 DK090971 (YZ).

1.. Langsigtet In vitro Erythropoiesis Kultur (Ex vivo erytroid Differentiering Culture protokol ved Giarratana et al. 8. Ændret og tilpasset til museceller) Dette er en 3-trins langsigtede in vitro erythropoiese protokol. I det første trin (dage 0-4) 2 x 10 5 celler / ml anbringes i erythroblastslægtskolonierne vækstmedium suppleret med stamcellefaktor (SCF), interleukin-3 (IL-3) og erythropoietin (Epo). I det andet trin (dage 5-6)...

<ol>
	<li>McGrath, K. E., Kingsley, P. D., Koniski, A. D., Porter, R. L., Bushnell, T. P., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+primitive+erythroid+cells+generates+a+transient+population+of+&amp;#34;pyrenocytes&amp;#34;+in+the+mammalian+fetus.">Enucleation of primitive erythroid cells generates a transient population of &amp;#34;pyrenocytes&amp;#34; in the mammalian fetus.</a> Blood. 111, 2409-2417 (2008).</li><li>Chasis, J. A., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythroblastic+islands:+niches+for+erythropoiesis.">Erythroblastic islands: niches for erythropoiesis.</a> Blood. 112, 470-478 (2008).</li><li>Koury, S. T., Koury, M. J., Bondurant, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+distribution+and+function+during+the+maturation+and+enucleation+of+mammalian+erythroblasts.">Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts.</a> J Cell Biol. 109, 3005-3013 (1989).</li><li>Ji, P., Jayapal, S. R., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+cultured+mouse+fetal+erythroblasts+requires+Rac+GTPases+and+mDia2.">Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2.</a> Nat Cell Biol. 10, 314-321 (2008).</li><li>Keerthivasan, G., Small, S., Liu, H., Wickrema, A., Crispino, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+trafficking+plays+a+novel+role+in+erythroblast+enucleation.">Vesicle trafficking plays a novel role in erythroblast enucleation.</a> Blood. 116, 3331-3340 (2010).</li><li>McGrath, K. E., Bushnell, T. P., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+hematopoietic+cells:+where+flow+meets+morphology.">Multispectral imaging of hematopoietic cells: where flow meets morphology.</a> J Immunol Methods. 336, 91-97 (2008).</li><li>Konstantinidis, D. G., 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=Signaling+and+cytoskeletal+requirements+in+erythroblast+enucleation.">Signaling and cytoskeletal requirements in erythroblast enucleation.</a> Blood. 119, 6118-6127 (2012).</li><li>Giarratana, M. 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=Ex+vivo+generation+of+fully+mature+human+red+blood+cells+from+hematopoietic+stem+cells.">Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells.</a> Nat Biotechnol. 23, 69-74 (2005).</li><li>Chen, K., Liu, J., Heck, S., Chasis, J. A., An, X., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+distinct+stages+in+erythroid+differentiation+based+on+dynamic+changes+in+membrane+protein+expression+during+erythropoiesis.">Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.</a> Proc Natl Acad Sci U S A. , (2009).</li><li>Kalfa, T. 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=Rac1+and+Rac2+GTPases+are+necessary+for+early+erythropoietic+expansion+in+the+bone+marrow+but+not+in+the+spleen.">Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen.</a> Haematologica. 95, 27-35 (2010).</li><li>Koulnis, M., Pop, R., Porpiglia, E., Shearstone, J. R., Hidalgo, D., Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+analysis+of+mouse+erythroid+progenitors+using+the+CD71/TER119+flow-cytometric+assay.">Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay.</a> J Vis Exp. , (2011).</li><li>Yoshida, H., Kawane, K., Koike, M., Mori, Y., Uchiyama, Y., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylserine-dependent+engulfment+by+macrophages+of+nuclei+from+erythroid+precursor+cells.">Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells.</a> Nature. 437, 754-758 (2005).</li><li>Ortyn, W. 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I de seneste år har studiet af erythroblast enucleation har vundet stigende fart, da det er det trin i in vitro erythropoiese kulturer, der er mest vanskelige at reproducere effektivt for at opnå en vellykket produktion af røde blodlegemer ex vivo i stor skala. Indtil for nylig undersøgelse af erythroblast enucleation udnyttes hovedsageligt fluorescensmikroskopi og flowcytometri metoder. Fluorescensmikroskopi metoder, omend nyttige i at identificere de deltagende molekyler kræve dages mikroskopisk...

Terminal differentiering i erythroid afstamning i pattedyr slutter med den dramatiske proces enucleation, hvorigennem orthochromatic erythroblastslægtskolonierne ekskluderer sin membran-indkapslet kerne (pyrenocyte) 1, genererer en reticulocyt 2. Den nøjagtige mekanisme af denne proces, som også er det hastighedsbegrænsende trin for vellykket stor skala produktion af røde blodlegemer in vitro, endnu ikke er fuldt belyst. Lokaliseringen af vigtige proteiner og strukturer inden enucleating Erytroblaster bygger på anvendelsen af fluorescerende og elektronmikroskopi 3-5. Denne kedelig proces resulterer typisk i identifikationen af ​​et begrænset antal enucleation begivenheder, og ikke altid tillader meningsfuld statistisk analyse. Udvidelse på en metode til erythroblast identifikation tidligere beskrevet af McGrath et al. 6, har vi udviklet en ny tilgang til at identificere og studere enucleation begivenheder ved Multi-Spectral ImaGing Flowcytometri (multiparameter high-speed cell imaging i flow, en metode, der kombinerer fluorescerende mikroskopi med flowcytometri) 7, som kan give et tilstrækkeligt antal observationer til at opnå målinger og statistisk analyse. Her beskriver vi to første in vitro erytropoiese kultur metoder, der anvendes med henblik på at synkronisere erytroblaster og øge sandsynligheden for at opfange enucleation på tidspunktet for evalueringen. Så vi beskriver i detaljer farvning af erytroblaster efter fiksering og permeabilisering for at studere lokalisering af intracellulære proteiner eller lipidklumper under enucleation af multi-spektral billeddannelse flowcytometri. Prøverne køres på en imaging flowcytometer og de ​​indsamlede celler gated hensigtsmæssigt at identificere orthochromatic erythroblaster 6. Orthochromatic erytroblaster analyseres derefter på grundlag af deres skærmformat, som målt i lysfelt imaging versus deres værdi for parameteren delta centroid XY TER119-DNA, som er defineret som afstanden mellem centrene af de områder, farvet for TER119 og DNA, hhv. Den population af celler med lav aspect ratio og høj delta tyngdepunkt XY Ter119/DNA er stærkt beriget i enucleating celler. Brug vildtype (WT) erythroblaster versus erytroblaster med MX-Cre medieret betinget sletning af Rac1 på Rac2 - / - eller kombineret Rac2 - / -; Rac3 - / - genetisk baggrund, og denne roman analyse protokol af multi-spektral billeddannelse flowcytometri, vi for nylig vist, at enucleation ligner asymmetrisk cytokinese og at dannelsen af en actomyosin ring reguleres delvist af Rac GTPaser er vigtigt for enucleation progression 7.

<table border="0" cellpadding="0" cellspacing="0" width="934">
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">&#945;MEM medium</td>
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		<tr height="29">
			<td height="29" style="height:29px;width:363px;">IMDM medium</td>
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			<td height="24" style="height:24px;width:363px;">AF594-cholera toxin B subunit</td>
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		<tr height="24">
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Ferric nitrate</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:207px;">F3002</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">EDTA</td>
			<td style="width:199px;">Fisher Scientific</td>
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		<tr height="27">
			<td height="27" style="height:27px;width:363px;">24-well plates</td>
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			<td style="width:167px;"></td>
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		<tr height="31">
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Syringe needle 25G5/8</td>
			<td style="width:199px;">BD</td>
			<td style="width:207px;">305122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Capped flow tubes</td>
			<td style="width:199px;">BD</td>
			<td style="width:207px;">352058</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:363px;">40&#956;m cell strainer</td>
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			<td style="width:207px;">352340</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Scalpel (disposable)</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
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			<td style="width:207px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
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			<td style="width:207px;"></td>
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		<tr height="48">
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			<td style="width:207px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:363px;">Hemavet 950 Cell Counter</td>
			<td style="width:199px;">Drew Scientific</td>
			<td>CDC-9950-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">NAPCO series 8000WJ Incubator</td>
			<td style="width:199px;">Thermo scientific</td>
			<td style="width:207px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Allegra X-15R Centrifuge</td>
			<td style="width:199px;">Beckman Coulter</td>
			<td>392932</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Mini Mouse Bench centrifuge</td>
			<td style="width:199px;">Denville</td>
			<td style="width:207px;">C0801</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

identification of a murine erythroblast subpopulation  enriched in enucleating events by multi-spectral imaging flow cytometry

Erythropoiese i pattedyr slutter med den dramatiske proces enucleation, der resulterer i reticulocyt-formation. Mekanismen af ​​enucleation er endnu ikke fuldt belyst. Et fælles problem, når man studerer lokalisering af vigtige proteiner og strukturer inden enucleating erytroblaster ved mikroskopi er vanskeligheden ved at observere et tilstrækkeligt antal celler undergår enucleation. Vi har udviklet en ny analyse protokol hjælp multiparameter højhastigheds-cell imaging i flow (Multi-Spectral Imaging Flowcytometri), en metode, der kombinerer immunofluorescent mikroskopi med flowcytometri, for at identificere effektivt et betydeligt antal enucleating begivenheder, der gør det muligt at få målinger og statistisk analyse. Vi først beskrive her to in vitro erythropoiese dyrkningsfremgangsmåder anvendes for at synkronisere murine Erytroblaster og øge sandsynligheden for at fange enucleation på the tid for evaluering. Derefter, vi beskriver i detaljer, farvning af erytroblaster efter fiksering og permeabilisering for at studere lokalisering af intracellulære proteiner eller lipidklumper under enucleation af multi-spektral billeddannelse flowcytometri. Sammen med størrelse og DNA/Ter119 farvning, som anvendes til at identificere de orthochromatic Erytroblaster anvender vi parametrene &quot;aspect ratio&quot; af en celle i den lyse felt kanal, der hjælper i anerkendelsen af ​​aflange celler og &quot;delta tyngdepunkt XY Ter119/Draq5 &quot;, der muliggør identifikation af cellulære begivenheder, hvor centrum af TER119 farvning (spirende reticulocyt) er langt fra centrum af DRAQ5 farvning (kerne undergår ekstrudering), hvilket indikerer således en celle ved at enucleate. Den delmængde af orthochromatic erythroblast befolkning med høj delta tyngdepunkt og lav skærmformat er stærkt beriget i enucleating celler.

Først analyseres cellerne baseret på deres Brightfield Aspect Ratio (forholdet mellem længden af ​​deres ringe versus deres hovedakse), og deres Brightfield Area (betegnende for deres størrelse). Begivenheder med en Brightfield Area-værdi lavere end 20, og over 200 er for det meste snavs og celleaggregater henholdsvis og er udelukket fra analysen (figur 2A). Enkelte celler (gate &quot;R1&quot;) er derefter analyseret på grundlag af deres værdi for Gradient RMS parameter, hvilket indikerer ska...

Watch this Scientific Journal Video about Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry at JoVE.com

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Den foreliggende protokol beskriver en hidtil ukendt fremgangsmåde til identificering af en population af enucleating orthochromatic erytroblaster af multi-spektral billeddannelse flowcytometri, hvilket giver en visualisering af erythroblast enucleation processen.

Identifikation af en murine erythroblast delpopulation beriget Enucleating Begivenheder af Multi-spektral Imaging Flowcytometri

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Research

JoVE Journal

Biology

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Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

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Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

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Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...