Diese Arbeit wurde teilweise durch CSIC (I + D Projekt C022) und PEDECIBA unterstützt. Die Autoren wollen Mariela Bollati und Valentina Porro aus der Zellbiologie Unit (Institut Pasteur de Montevideo) danken für die großzügige Zusammenarbeit über die MoFlo Zellsortierer und Merial-Montevideo für sanft bietet alle Meerschweinchen Proben in diesem Projekt verwendet. Abbildung 1 Abbildungen 2 und 3 und Tabelle 1 mit Genehmigung der S. Karger AG, Basel;; wurde mit freundlicher Genehmigung von BioMed Central reproduziert und die Abbildungen 4 und 5 wurden reproduziert oder mit Genehmigung von John Wiley &amp; Sons, Inc. (Copyright im Besitz angepasst Wiley- Blackwell, 2011).

1. Herstellung von Zellsuspensionen <ol><li> Sacrifice die Probe entsprechend den Empfehlungen der Fachausschüsse wie IACUC oder gleichwertig verwendet werden (in Uruguay, Nationale Kommission für Tierversuche [CNEA]). In unserem Fall wurde eine Überdosis Pentobarbital verabreicht. </li><li> Präparieren Sie die Hoden nach der Norm zulässigen Verfahren und legen Sie sie in einem 96 mm Glas Petrischale auf Eis, mit 10 ml eiskaltem DMEM ergänzt mit 10% fötalem Kälberserum. </li><li> Entfernen Sie die Tunica albuginea und schneiden Sie die entkapselten Hoden in quadratische Stücke von 2 - 3 mm auf jeder Seite. </li><li> Diese Stücke werden dann in einem Medimachine eine automatisierte mechanische Mühle, wo das Gewebe in einer austauschbaren Einheit, die eine perforierte Edelstahlsieb und ein Metall Rotor aufgeschlüsselt verarbeitet wird. 5 dieser Stücke in einem 50 um Einweg-Einheit, Schalter auf der disaggregator und Prozess - Um dies zu tun, Platz 1 ml kaltem DMEM und 4 ergänzt<html> für 50 Sekunden die folgenden, einfachen Anweisungen des Herstellers. </li><li> Wiederherstellen der resultierende Zellsuspension aus der Aufschlüsselung Gerät mit einer 3 - 5 ml Spritze ohne Nadel. </li><li> Man filtriert durch ein 50 um Nylon-Mesh, vorher eingeweicht mit 0,5 ml DMEM ergänzt. </li><li> Filtern Sie die Suspension wieder mit einem getränkten 25 um Nylon-Mesh und auf Eis stellen. </li><li> Zählen in einer Neubauer-Kammer und stellen zelluläre Konzentration auf 1 - 2 x 10 7 Zellen / ml mit DMEM ergänzt. Mindestens 4 x 10 7 Zellen / Gramm Hoden Material werden üblicherweise erhalten. </li><li> Schließlich, fügen NDA (2-naphthol-6 ,8-disulfonsäure Dikaliumsalz) bis zu einer Endkonzentration von 0,2%, um Zelle Verklumpung verhindern. </li><li> Optional: Überprüfen Sie die Lebensfähigkeit der Zellen der Hoden Zellsuspensionen mit einem kommerziell erhältlichen Kit Lebensfähigkeit für tierische Zellen, nach den Anweisungen des Herstellers. </li></ol> 2. Durchflusszytometrieanalyse ve_content &quot;&gt; Wir haben ein Becton-Dickinson FACSVantage Durchflusszytometer mit einer kohärenten Argon-Ionen-Laser Resonanz bei 488 nm für die Analyse von Zellen mit hohen Konzentrationen von Propidiumiodid (PI) gefärbt emittieren eingesetzt wird. ausgestellt (PI Eintritt in unfixierten Zellen unter Stress wurde an anderer Stelle 9,10) gerichtet. <ol><li> Für PI-Färbung, fügen Fluorochrom in einer Endkonzentration von 50 ug / ml der Zellsuspension, und Inkubation für 10 min bei 0 ° C in der Dunkelheit. </li><li> Laserleistung bis 100 mW und einen 575/26 Bandpass-Filter verwendet wird, um PI-emittierten Fluoreszenz in FL2 sammeln eingestellt. </li><li> Wir führen FC Messungen mit einem 70 um Düse. Für die Sortierung Spermatozyte Populationen gesetzt Sortierung Modus Normales-R oder normal-C, unter Verwendung von 3 sortiert Tropfen als Umschlag. Halten Sie Probe und Sammlung Röhrchen bei 3-4 ° C unter Verwendung einer Kühleinheit. Einstellen Probe Differential an Zellen mit einer Geschwindigkeit von 500 bis 1500 pro Sekunde zu analysieren. </li><li> Verwenden CellQuest Software (BD) zu analysieren,die folgenden Parameter: forward scatter (FSC-H); Seitwärtsstreuung (SSC-H); insgesamt emittierten Fluoreszenz-oder Puls-Bereich (FL2-A) und Dauer der Fluoreszenz-Emission oder Puls-Weiten (FL2-W). </li></ol> Alternativ haben wir die vitalen Farbstoff Hoechst 33342 in einer Endkonzentration von 5 ug / ml eingesetzt und inkubiert 10 min bei 37 ° C im Dunkeln. Zelle Analyse wurde mittels eines MoFlo Zytometer (DakoCytomation), ausgestattet mit einem UV-Anregungswellenlänge Laser, der bei 25 mW und mit einer Düse 70 um erfolgt. Datenmanipulation wurde mit Summit v4.3 Software (DakoCytomation) durchgeführt.

<ol>
	<li>Meistrich, M. L., Prescott, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+spermatogenic+cells+and+nuclei+from+rodent+testes.">Separation of spermatogenic cells and nuclei from rodent testes.</a> Methods of Cell Biology. 15, 15-54 (1977).</li><li>Lam, D. M. K., Furrer, R., Bruce, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+separation,+physical+characterization,+and+differentiation+kinetics+of+spermatogonial+cells+of+the+mouse.">The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse.</a> Proc. Natl. Acad. Sci. USA. 65, 192-199 (1970).</li><li>Romrell, L. J., Bellve, A. R., Fawcet, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.">Separation of mouse spermatogenic cells by sedimentation velocity.</a> Dev. Biol. 19, 119-131 (1976).</li><li>Geisinger, A., Rodr&#237;guez-Casuriaga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+for+gene+expression+studies+in+mammalian+spermatogenesis.">Flow cytometry for gene expression studies in mammalian spermatogenesis.</a> Cytogenet. Genome Res. 128, 46-56 (2010).</li><li>Getun, I. V., Torres, B., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. (50), e2602 (2011).</li><li>Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+velocity+sedimentation.">Separation of mouse spermatogenic cells by velocity sedimentation.</a> J. Cell Physiol. 80, 299-312 (1972).</li><li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Malkov, M., Fisher, Y., Don, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+schedule+of+the+postnatal+rat+testis+determined+by+flow+cytometry.">Developmental schedule of the postnatal rat testis determined by flow cytometry.</a> Biol. Rep. 59, 84-92 (1998).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., Santi&#241;aque, F., L&#243;pez, B., Folle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-purity+flow+sorting+of+early+meiocytes+based+on+DNA+analysis+of+guinea+pig+spermatogenic+cells.">High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.</a> Cytometry A. 79, 625-634 (2011).</li><li>Davey, H. M., Hexley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+but+not+dead?+Membranes+of+stressed+Saccharomyces+cerevisiae+are+permeable+to+propidium+iodide.">Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide.</a> Environ. Microbiol. 13, 163-171 (2011).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., L&#243;pez-Carro, B., Porro, V., Wettstein, R., Folle, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-fast+and+optimized+method+for+the+preparation+of+rodent+testicular+cells+for+flow+cytometric+analysis.">Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.</a> Biol. Proced. Online. 11, 184-195 (2009).</li><li>Span&#242;, M., Evenson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+for+reproductive+biology.">Flow cytometric analysis for reproductive biology.</a> Biol Cell. 78, 53-62 (1993).</li></ol>

Die optimierte hier beschriebene Verfahren ermöglicht die Herstellung von Zellsuspensionen aus Nagetier Hodengewebe in einem sehr schnellen und reproduzierbaren Weise vermeiden Enzym und Reinigungsmittel Behandlung und das Beibehalten der guten Integrität der Zelle und Art Proportionen. Die Kürze des Verfahrens (die 15 min Spannweite umfasst Hoden Dissektion, Schneiden von Gewebe und Verarbeitung), Aufwand ist minimal, und das Fehlen von enzymatischen Behandlungen sind nur einige der wichtigsten Vorteile. All dies w?...

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Medimachine system</td>
 <td>BD</td>
 <td>340587</td>
 <td></td>
 </tr>
 <tr>
 <td>Medicon unit (50 &mu;m)</td>
 <td>BD</td>
 <td>340591</td>
 <td></td>
 </tr>
 <tr>
 <td>Filcon unit (50 &mu;m)</td>
 <td>BD</td>
 <td>340603</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>430-2100</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>PAA</td>
 <td>A11-151</td>
 <td></td>
 </tr>
 <tr>
 <td>NDA</td>
 <td>Chemos BmbH</td>
 <td>277081</td>
 <td></td>
 </tr>
 <tr>
 <td>Hoechst 33342</td>
 <td>Sigma-Aldrich</td>
 <td>14533</td>
 <td>Stock solution 5 mg/ml; used at a final concentration of 5 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Propidium iodide</td>
 <td>Sigma-Aldrich</td>
 <td>287075</td>
 <td>Stock solution 1 mg/ml; used at a final concentration of 50 &mu;g/ml</td>
 </tr>
 </tbody>
</table>

simple and efficient technique for the preparation of testicular cell suspensions

Mammalian Hoden sehr komplexe Organe, die mehr als 30 verschiedenen Zelltypen, einschließlich somatischen Zellen und Hoden verschiedenen Stadien der Keimbahn-Zellen enthalten. Diese Heterogenität ist ein wichtiger Nachteil bezüglich der Studie von den Basen der Säuger Spermatogenese, als reiner oder angereicherter Zellpopulationen in bestimmten Stadien der Entwicklung der Spermien für die meisten molekularen Analysen 1 benötigt. Verschiedene Strategien wie Staput 2,3, zentrifugale Elutriation 1 und Durchflusszytometrie (FC) 4,5 wurden eingesetzt, um angereichert oder gereinigt Hoden Zellpopulationen zu erhalten, um differentiellen Genexpression Studien zu ermöglichen. Es ist erforderlich, dass die Zellen in Suspension für die meisten Anreicherung / Reinigung möglich sind. Im Idealfall wird die Zellsuspension werden Vertreter des ursprünglichen Gewebes, haben einen hohen Anteil an lebensfähigen Zellen und nur wenige multinucleates - die dazu neigen, wegen der Form,Syncytial Natur des Samenepithels 6,7 - und der Mangel Zellklumpen 1. Frühere Berichte hatten bewiesen, dass Hoden Zellsuspensionen von einem ausschließlich mechanischen Verfahren hergestellt leichter verklumpt als diejenigen trypsinierten 1. Auf der anderen Seite, enzymatische Behandlungen mit RNasen und / oder Disaggregation Enzyme wie Trypsin und Collagenase führen zu Makromolekülen Abbau, was für die bestimmten Downstream-Anwendungen ist. Der ideale Prozess sollte so kurz wie möglich und beinhalten minimale Manipulation, um so eine gute Erhaltung der Makromoleküle von Interesse, wie mRNAs zu erreichen. Diese Protokolle zur Herstellung von Zellsuspensionen aus festen Geweben sind in der Regel zeitaufwendig, sehr bedienerabhängige und wahlweise beschädigen bestimmten Zelltypen 1,8. Das Protokoll hier vereint die Vorteile einer hoch reproduzierbar und extrem kurzen mechanischen Aufschluss mit dem einbsence der enzymatischen Behandlung, was eine gute Qualität Zellsuspensionen, die für die durchflusszytometrische Analyse und Sortierung 4 und anderweitige Genexpressionsstudien 9 verwendet werden kann.

Ein Beispiel für ein gut aufgeschlüsselten Zellsuspension aus Rattenhoden mit dem hier beschriebenen Protokoll ist in Abbildung 1 dargestellt. Im Vergleich zur enzymatischen Behandlungen 6,8 und den zuvor beschriebenen mechanischen Aufschluss Verfahren 2 ist die hier vorgestellte viel schneller beinhaltet weniger Handhabung ist leicht reproduzierbar (nicht bedienerabhängige) und macht knapp Zelltrümmer (insbesondere im Vergleich zu anderen mechanisc...

Watch this Scientific Journal Video about Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions at JoVE.com

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Ein neuartiges Protokoll für die mechanische Aufbereitung von Hoden Zellsuspensionen von Nagetier Material, die Vermeidung Enzyme und Reinigungsmittel, beschrieben. Das Verfahren ist sehr einfach, schnell, reproduzierbar und macht gute Qualität Zellsuspensionen, die sich für Durchfluss Sortier-und RNA-Extraktion sind.

Einfache und effiziente Technik zur Herstellung von Hodenkrebs Zellsuspensionen

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

simple-efficient-technique-for-preparation-testicular-cell

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Biology

18.6K Aufrufe.  Instituto de Investigaciones Biológicas Clemente Estable (IIBCE). Ein neuartiges Protokoll für die mechanische Aufbereitung von Hoden Zellsuspensionen von Nagetier Material, die Vermeidung Enzyme und Reinigungsmittel, beschrieben. Das Verfahren ist sehr einfach, schnell, reproduzierbar und macht gute Qualität Zellsuspensionen, die sich für Durchfluss Sortier-und RNA-Extraktion sind.

Serie: Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Erstellung von Einzel-Zellsuspensionen aus Maus-Milz mit dem gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Forschung

Biologie

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

Standardisierte Erstellung von Einzel-Zellsuspensionen aus Maus Lungengewebe mit dem gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Vorbereitung der gepoolten menschlichen Thrombozyten-Lysat (PHPL) als effiziente Ergänzung zum tierischen Serum-freien menschlichen Stammzell-Kulturen

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Erstellung von Qualitäts-Inositol Pyrophosphate

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Messung von pH Vacuolar und Cytosolic<em&gt; In Vivo</em&gt; In Hefe Zellsuspensionen

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Effiziente Agroinfiltration von Pflanzen für High-Level-Transient Expression von rekombinanten Proteinen

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

Folienvorbereitungsmethode zur Erhaltung der dreidimensionalen Chromatinarchitektur von Hodenkeimzellen

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Effiziente iPS Zellgeneration aus Blut Mit Episomen und HDAC-Inhibitoren

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Effiziente Mammalian Cell Expression und Single-step Reinigung der extrazellulären Glykoproteinen für die Kristallisation

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...