Forfatterne takker Forsknings flowcytometrisystemer Kjerne ved Cincinnati Children Hospital Research Foundation og Richard Demarco, Sherree venn, og Scott Mordekai fra Amnis Corporation (del av EMD Milllipore) for ekspert teknisk support. Dette arbeidet ble støttet av National Institutes of Health gir K08HL088126 og R01HL116352 (TAK) og P30 DK090971 (YZ).

1. Langsiktig In vitro Erytropoese Culture (Ex vivo erythroid Differensiering Kultur Protocol etter Giarratana et al. 8, endret og tilrettelagt for Mus Cells) Dette er en 3-trinns langsiktig in vitro erytropoese protokollen. I det første trinnet (dager 0-4) 2 x 10 5 celler / ml er plassert i erytroblast vekstmedium supplert med stamcellefaktor (SCF), interleukin-3 (IL-3), erytropoietin (EPO). I det andre trinnet (5-6 dager), blir celle...

<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. 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I de senere år studiet av erytroblast enucleation har fått økende moment siden det er trinnet i in vitro erytropoiese kulturer som er mest vanskelig å reprodusere en effektiv måte for å oppnå vellykket, stor-skala produksjon av røde blodceller ex vivo. Inntil nylig studiet av erytroblast enucleation utnyttes hovedsakelig fluorescens mikroskopi og flowcytometri metoder. Fluorescensmikroskopi metoder, enskjønt nyttig for å identifisere molekyler som deltar, krever dager med mikroskopis...

Terminal differensiering innen erythroid avstamning i pattedyr konkluderer med den dramatiske prosessen med enucleation, gjennom hvilket ortokromatiske erytroblast expels sin membran-innkapslet nucleus (pyrenocyte) 1, genererer retikulocytter to. Den nøyaktige mekanisme for denne prosess, som også er det hastighetsbegrensende trinn i vellykket, i stor skala, produksjonen av røde blodceller in vitro, er ennå ikke helt klarlagt. Lokalisering av viktige proteiner og strukturer innenfor enucleating erytroblaster er avhengig av bruk av fluoriserende og elektronmikroskopi 3-5. Denne langtekkelig prosess vanligvis resulterer i identifisering av et begrenset antall enucleation hendelser og ikke tillater alltid menings statistisk analyse. Utvidelse på en metode for erytroblast identifikasjon beskrevet tidligere av McGrath et al. 6, har vi utviklet en ny tilnærming for å identifisere og studere enucleation hendelser av Multi-Spectral ImaGing flowcytometri (multiparameter høyhastighets celle bildebehandling i flyt, en metode som kombinerer fluorescerende mikroskopi med flowcytometri) 7, som kan gi et tilstrekkelig antall observasjoner til å foreta målinger og utføre statistisk analyse. Her beskriver vi første to in vitro erytropoiese kultur metoder som benyttes for å synkronisere erytroblaster, og øker sannsynligheten for å fange enucleation ved tidspunktet for evaluering. Da vi beskriver i detalj farging av erytroblaster etter fiksering og permeabilization for å studere lokalisering av intracellulære proteiner eller lipid flåter under enucleation ved multispektral avbildning strømningscytometri. Prøvene blir drevet på en bilde flowcytometer og de ​​innsamlede cellene er gated hensiktsmessig å identifisere ortokromatiske erytroblaster seks. Ortokromatiske erytroblaster blir deretter analysert basert på deres størrelsesforhold, som målt i lysfelt imaging, mot deres verdi for parameteren delta sentroide XY Ter119-DNA, som er definert som avstanden mellom sentrene av de områdene farget for Ter119 og DNA, henholdsvis. Befolkningen i celler med lav sideforhold og høy deltaet Tyngdepunktet XY Ter119/DNA er høyanriket i enucleating celler. Ved hjelp av wild-type (WT) erytroblaster versus erytroblaster med Mx-Cre mediert betinget sletting av Rac1 på Rac2 - / - eller kombinert Rac2 - / -; Rac3 - / - genetisk bakgrunn, og denne nye analyse protokoll for multispektral avbildning flowcytometri, vi nylig demonstrert at enucleation ligner asymmetrisk cytokinese og at dannelsen av en actomyosin ring reguleres delvis av Rac GTPases er viktig for enucleation progresjon 7..

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		<tr height="22">
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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">
			<td height="24" style="height:24px;width:363px;">pMRLC (Ser19) antibody</td>
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		<tr height="24">
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		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Ferrous sulfate</td>
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			<td style="width:167px;"></td>
		</tr>
		<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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">EDTA</td>
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			<td style="width:207px;">BP120500</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">
			<td height="31" style="height:31px;width:363px;">Flow tubes</td>
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			<td height="22" style="height:22px;width:363px;">Syringe needle 25G5/8</td>
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			<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>
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			<td style="width:207px;">352058</td>
			<td style="width:167px;"></td>
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		<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>
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		<tr height="22">
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			<td style="width:167px;"></td>
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		<tr height="22">
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			<td style="width:207px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:363px;">ImagestreamX Mark II Imaging Flow Cytometer</td>
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			<td style="width:207px;"></td>
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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

Erytropoese i pattedyr avsluttes med den dramatiske prosessen enucleation som resulterer i retikulocytt-formasjon. Mekanismen enucleation har ennå ikke fullstendig klarlagt. Et vanlig problem som oppstår når man studerer lokalisering av nøkkelproteiner og strukturer innenfor enucleating erytroblaster ved mikroskopi er vanskeligheten med å overholde et tilstrekkelig antall celler som gjennomgår enucleation. Vi har utviklet en ny analyse protokollen ved hjelp av multiparameter høyhastighets celle bildebehandling i flyt (Multi-Spectral Imaging flowcytometri), en metode som kombinerer immunofluorescent mikroskopi med flowcytometri, for å identifisere effektivt et betydelig antall enucleating hendelser, som gjør det mulig å å foreta målinger og utføre statistisk analyse. Vi først beskrive her to in vitro erytropoiese kultur metoder som brukes for å synkronisere murine erytroblaster og øke sannsynligheten for å fange enucleation på the tid for evaluering. Deretter vi beskriver i detalj farging av erytroblaster etter fiksering og permeabilization for å studere lokalisering av intracellulære proteiner eller lipid flåter under enucleation ved multispektral avbildning strømningscytometri. Sammen med størrelse og DNA/Ter119 flekker som brukes til å identifisere de ortokromatiske erytroblaster, vi utnytter de parameterne &quot;aspect ratio&quot; på en celle i den lyse-feltet kanal som hjelpemidler i erkjennelsen av langstrakte celler og &quot;delta Tyngdepunktet XY Ter119/Draq5 &quot;som gjør det mulig å identifisere cellulære hendelser der sentrum av Ter119 farging (begynnende retikulocytt) ligger langt fra hverandre fra midten av Draq5 farging (nucleus gjennomgår ekstrudering), noe som indikerer en celle om å enucleate. Undergruppe av den ortokromatiske erytroblast befolkning med høy delta Tyngdepunktet og lav sideforhold er høyanriket i enucleating celler.

Først blir cellene analysert basert på deres Lysfelt sideforhold (forholdet mellom lengden av deres mindre versus sin hovedakse) og deres Lysfelt området (som indikerer størrelsen). Hendelser med en Lysfelt Area-verdi lavere enn 20 og høyere enn 200 er det meste rusk og celleaggregater, henholdsvis, og er ekskludert fra analysen (Figur 2A). Enkeltceller (gate &quot;R1&quot;) er deretter analysert basert på deres verdi for Gradient RMS parameter, noe som indikerer skarphet i bildet. Gate &quot;R2&q...

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 protokoll beskriver en ny fremgangsmåte for identifisering av en populasjon av enucleating ortokromatiske erytroblaster ved multispektral avbildning flowcytometri, som gir en visualisering av erytroblast enucleation prosessen.

Identifikasjon av en Murint erytroblast undergruppe Beriket i Enucleating Hendelser etter Multi-spektral Imaging flowcytometrisystemer

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

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

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Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

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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 ...