Schwanzvene Injektionen von fluoreszierenden Farbstoffen Dextrane <ol><li> Um Bild zerebrale Gefäßsystem, müssen die Tiere intravenös mit einem fluoreszierenden Farbstoff injiziert werden. Wir verwenden Dextran-konjugierten Farbstoffe, weil die Dextran-Einheit verhindert der Farbstoff vor der Kreuzung das Gehirn Blut-Schranke und ein Auslaufen der Blutgefäße. </li><li> Mäuse sind mit Isofluran (4% für die Induktion, 1.5-2% während der Injektion). </li><li> Der Schwanz ist mit 70% Alkohol desinfiziert. </li><li> Mit einer 26-Gauge-Nadel, ist 75-100 ul einer 5% v / v Lösung von Rhodamin Dextran in Kochsalzlösung gelöst durch die Schwanzvene, auf halbem Weg entlang der Welle des Schwanzes injiziert. </li><li> Die Tiere können sofort nach der Schwanzvene Injektion abgebildet werden, bis der Farbstoff im Urin, was wiederum rosa wird, wenn mit einem Rhodamin-Farbstoffs in ca. 2 Stunden ausgeschieden wird. </li></ol> Imaging des Blutflusses mittels Zwei-Photonen-Mikroskopie (Gesamtdauer 30-60 min pro Sitzung, abhängig von der Zahl der Schiffe abgebildet) <ol><li> Nach dem fluoreszierenden Farbstoff Dextran Injektion wird die Maus mit Isofluran (4% für die Induktion, 1-1,5% für die Bildgebung). </li><li> Die Maus ist fest verbunden mit dem Titan-bar an das Mikroskop Bühne, die eine thermo-regulierten Heizkissen, damit die Tier warm enthält. Einige Augensalbe angewendet wird, damit die Augen feucht. </li><li> Das Deckglas der Schädelbasis Fenster ist mit 70% igem Alkohol gereinigt. </li><li> Das Fenster ist parallel zur Bildebene positioniert und zentriert auf dem Gebiet der Blick unter die 4X Ziel. </li><li> Am besten ist es, ein Foto von der Hirnoberfläche Schiffe mit einer digitalen Kamera aufnehmen. Dieses Bild wird als Bild der Referenz in folgenden Imaging-Sitzungen, um das abgebildete Region immer wieder zu finden von Tag zu Tag verwendet werden. </li><li> Die 4X Ziel ist es, mit dem Eintauchen in Wasser 40X-Objektiv ohne Verschieben der Bühne ersetzt. Ein digitales Bild des Sichtfeldes wird wieder aufgenommen. Die Koordinaten auf der Bühne Manipulator sind auf Null gesetzt. </li><li> Wir verwenden scanimage die Bildaufnahme-Software. Dies wurde in MatLab von Tom Pologruto und Bernardo Sabatini im Labor von Karel Svoboda (Pologruto et al., 2003) geschrieben. Laser, Leistungsmesser, der Photomultiplier Röhren, Verstärker, etc.: Wir als nächstes auf alle anderen Geräte wieder </li><li> Der Bereich des Fensters geeignet, die abgebildet werden soll, kurz bei geringer Vergrößerung gescannt, um die besten Regionen zu finden. Sobald diese identifiziert sind, eine geringe Vergrößerung Stapel von der gewählten Region um Bild aufgenommen wird, und dessen XY-Koordinaten sind annotiert. </li><li> Zur Aufzeichnung des Blutflusses Dynamik in kleinen Gefäßen oder Kapillaren verwenden wir Linien-Scans entlang mindestens 40 mu m des Schiffes von Interesse. Diese einzelnen &quot;Sweeps&quot; dauerhafte 1 Sekunde erfolgen mit so wenig Laserleistung wie möglich und die Koordinaten und Winkel des Scannens werden aufgeschrieben. </li><li> Sobald die Bildgebung Sitzung vorbei ist, ist das Tier in einen warmen Raum, in dem sie aus der Narkose erholen können verschoben. </li></ol>

<ol>
	<li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proc Nat Acad Sci. 95, 15741-15746 (1998).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed. Eng. Online. 17, 2-13 (2003).</li><li>Zhang, S., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+impact+of+cortical+microcirculation+on+synaptic+structure+and+sensory-evoked+hemodynamic+responses+in+vivo.">Imaging the impact of cortical microcirculation on synaptic structure and sensory-evoked hemodynamic responses in vivo.</a> 5, (2007).</li><li>Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrating+arterioles+are+a+bottleneck+in+the+perfusion+of+neocortex.">Penetrating arterioles are a bottleneck in the perfusion of neocortex.</a> Proc Nat Acad Sci. 104, 365-370 (2007).</li></ol>

Kortikale Durchblutung Dynamik kann in vivo durch bildgebende fluoreszierenden Dextran Farbstoffe in die Schwanzvene von Nagetieren, die mit Zwei-Photonen-Mikroskopie injiziert untersucht werden. Dieses Video zeigte eine Methode, wie Bild Blutfluss Dynamik in Neokortex der Maus über einen mit Glas überdachten kranialen Fenster Vorbereitung.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fluorescein isothio-cyanate-dextran</td>
<td>Reagent</td>
<td>Sigma</td>
<td>FD500S</td>
<td>mol wt 500,000</td>
</tr>
<tr>
<td>Rhodamine B isothio-cyanate-dextran</td>
<td>Reagent</td>
<td>Simga</td>
<td>R9379</td>
<td>mol wt ~70,000
</td>
</tr>		</tbody>
		</table>
		</div>

a method for 2-photon imaging of blood flow in the neocortex through a cranial window

Die Fähigkeit zur Abbildung der zerebralen Gefäße (von großen Schiffen auf Kapillaren) und Aufzeichnung des Blutflusses Dynamik im intakten Gehirn von lebenden Nagern ist eine leistungsstarke Technik. Mit In-vivo-2-Photonen-Mikroskopie durch ein Schädel-Fenster ist es möglich, Bild fluoreszierende Farbstoffe intravenös injiziert. Dies erlaubt es, Bild der kortikalen Gefäße und auch Messungen des Blutflusses erhalten. Diese Technik wurde ursprünglich von David Kleinfeld und Winfried Denk entwickelt. Die Methode kann verwendet werden, um den Blutfluss Dynamik während oder nach der zerebralen Ischämie zu studieren, in neurodegenerativen Erkrankungen, bei Hirntumoren oder in normale Physiologie des Gehirns. Zum Beispiel wurde verwendet, um zu untersuchen, wie Schlaganfall verursacht Veränderungen in den Blutfluss Richtung und Veränderungen in der roten Blutkörperchen Geschwindigkeit oder Fluss in und rund um den Infarkt. Hier zeigen wir, wie 2-Photonen-Mikroskopie zum Bild des Blutflusses Dynamik den Einsatz in der Großhirnrinde von Mäusen mit fluoreszierenden Farbstoffen in die Schwanzvene injiziert.

Watch this Scientific Journal Video about A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window at JoVE.com

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

Kortikale Durchblutung Dynamik kann in vivo durch bildgebende fluoreszierenden Dextran Farbstoffe in die Schwanzvene von Nagetieren, die mit 2-Photonen-Mikroskopie injiziert untersucht werden. Dieses Video zeigt, wie man Bild Blutfluss Dynamik in Neokortex der Maus über einen mit Glas überdachten kranialen Fenster Vorbereitung.

Eine Methode zur 2-Photonen-Imaging der Blutfluss in den Neocortex durch ein Cranial Fenster

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

a-method-for-2-photon-imaging-blood-flow-neocortex-through-cranial

Research

JoVE Journal

Biology

18.7K Aufrufe.  University of California, Los Angeles. Kortikale Durchblutung Dynamik kann in vivo durch bildgebende fluoreszierenden Dextran Farbstoffe in die Schwanzvene von Nagetieren, die mit 2-Photonen-Mikroskopie injiziert untersucht werden. Dieses Video zeigt, wie man Bild Blutfluss Dynamik in Neokortex der Maus über einen mit Glas überdachten kranialen Fenster Vorbereitung.

Serie: A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Dissection und 2-Photonen-Imaging der peripheren Lymphknoten in Mäusen

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Forschung

Biologie

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Eine einfache Methode für genaue Genotypisierung

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

In situ Imaging der Maus Thymus mit 2-Photonen-Mikroskopie

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

In Vivo 2-Photon Calcium Imaging in Layer 2 / 3 der Mäuse

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

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.

Generierung von Knochenmark murinen dendritischen Zellen für den Einsatz in 2-Photonen-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% ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

Verwendung von Drehstabtest als Methode für die qualitative Analyse von Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

Dendra2 Photoschalten durch die Mammary Imaging Fenster

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Eine einfache Methode zur Bildgebung Arabidopsis Leaves Mit Perfluordecalin als Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Eine Explantation Assay zur Beurteilung Cellular Verhalten des Cranial Mesenchym

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

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

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Identifizierung einer Subpopulation Murine Erythroblast Enriched in Enucleating Veranstaltungen durch multispektrale Imaging Durchflusszytometrie

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