1. Vorbereitung der Proben <ol><li> Verdünnen der Zellsuspension mit ORFLO Verdünnungsmittel oder einem geeigneten Verdünnungsmittel, so daß die Zellkonzentration im Arbeitsbereich des Instruments (3.000 bis 500.000 Zellen / ml für den Typ M-Kassette, oder 3.000 bis 2.500.000 Zellen / ml für Typ S-Kassette) ist. </li><li> Eine Verdünnung von 1:5 bis 1:20 (Typ M-Kassette) oder ohne Verdünnung bis 1:5 Verdünnung (Typ S-Kassette) ist für die meisten Säugetier-Zelllinien empfohlen, aber die entsprechende Verdünnung wird nach Zelltyp und Aussaatdichte abhängen. Das Volumen für eine genaue Zählung erforderlich ist ungefähr 75 ul. </li></ol> 2. Die Proben laufen <ol><li> Drehen Sie das Moxi Z durch Drücken der Power-Taste und die Home-Bildschirm wird angezeigt. </li><li> Drücken Sie das Tablett ab und legen Sie eine Kassette in das Moxi Z. Die Pipette 75μL Probe ... wird angezeigt. </li><li> Pipettieren Sie 75 &amp;mgr; l Probe in die Einfüllöffnung der Kassette (blaues Ovalbeschriftet 1 oder 2, je nachdem, welcher Ende der Kassette wurde in das Gerät eingesetzt). Dies sind die Einweg-Kassetten, die für 2 Tests verwendet werden können. Hinweis: Setzen Sie die Pipettenspitze direkt in den Laden gut bei ~ 45 °-Winkel, so dass die Spitze unter der vorderen Lippe der gut gefangen wird. Beim Dosieren, ist es wichtig, dass das Vakuum nicht &quot;weiterkommen&quot; der Abgabe der Flüssigkeit wie Lufteinschlüsse verursachen könnten, um die Kassette geben. Es wird empfohlen, eine kleine Kugel (die Größe der Eintrag well) flüssiger Form über die auch während der Abgabe haben. </li><li> Zum Zählen meisten Säugetierzellen, berühren Sie den Bildschirm irgendwo zu beginnen. Für die Zählung sehr kleiner Teilchen (4 bis 8 um mittlere Durchmesser für Typ M und um 08.03 durchschnittlichen Durchmesser für Typ S), kann die Moxi Z in Small Particle-Modus ausgeführt werden. Hinweis: die Kassette Typ wird in der schwarzen Leiste am oberen Rand des Bildschirms angezeigt. In diesem Modus setzt Moxi Z den Durchmesser Skala auf 2 bis 10 um als Standard und führt die Anzahl usingen optimierten Parameter für den Nachweis von kleinen Zellen. Drücken Sie die TOUCH HIER Small Particle Mode-Taste (schwarzes Quadrat), um den Test zu starten und laufen in diesem Modus. </li><li> Das Moxi Z beginnen Sie den Test und die Zellzahl Ergebnisse werden in ca. 8 Sekunden (Typ M-Kassette) oder 15 Sekunden (Typ S-Kassette) abgeschlossen. </li><li> Die Curve Fitting und MVI Berechnungen beginnen automatisch und erfordert nur ein paar zusätzliche Sekunden. Wenn ein geeigneter Sitz gefunden wird, werden die Ergebnisse dann automatisch auf dem Bildschirm angezeigt werden. </li><li> Um den Standard-Gating Erwerb Modus vorzunehmen, drücken Sie die Kurvenanpassung Graf-Taste, um in Gating-Modus umzuschalten. </li><li> An diesem Punkt kann die Kassette aus dem Gerät entfernt werden. </li></ol> 3. Verwalten von Daten <ol><li> Wenn AutoMode Skalierung ausgeschaltet ist, werden die Ergebnisse zunächst für eine Kurvenanpassung Zählung auf einer Skala von 2-34 Durchmesser um (Typ M-Kassette) oder 2-26 μ angezeigtm (Typ S-Kassette). Wenn Automode Skalierung ist, dann wird die Moxi Z automatisch in die Skala der horizontalen Achse (x-Achse) auf den Bereich, der der Breite der Kurvenanpassung Bevölkerung am nächsten ist und gleichzeitig alle Kurvenanpassung Daten angezeigt werden. </li><li> Wenn AutoMode Skalierung ausgeschaltet ist, um manuell die Ergebnisse mit einer höheren Auflösung, berühren Sie das Symbol um 26.02 (Typ M) oder das Symbol um 18.02 (Typ S). Hinweis: Die Kassette Typ wird in dem blauen Feld oben links auf dem Bildschirm angezeigt. Dies wird empfohlen, beim Zählen kleinere Zellen oder Partikel, sowohl die Kurvenanpassung Fähigkeiten sowie die Fähigkeit des Benutzers, um visuell zu unterscheiden Nuancen der Daten zu verbessern. </li><li> Bei Erhöhung des horizontalen Auflösung, wird die Schaltfläche Skalierung dynamisch anpassen, um für das Radfahren zwischen den zur Verfügung stehenden x-Achse Skala reicht erlauben: 2-34, 2-26, 2-18 und 2-10 um für den Typ M-Kassette; oder 2 -26, 18.02, 10.02 und um für die Type S-Kassette. Diese Funktion ist nur AvailaZweidrahtleitung unmittelbar nach einer Zellzahl. </li><li> Zählen Sie Informationen (Zellen / ml) für die Kurve-fit oder gated Region ist in einem schwarzen Kasten unter der linken Seite des Histogramms angezeigt. Mittlere Zellgröße beruhen in dem schwarzen Kasten unter der rechten Seite des Histogramms angezeigt. </li><li> Der MVI-Wert ist ein blaues Feld oben rechts im Histogramm angezeigt. Wenn der Test in kleinen Partikel-Modus ausgeführt wurde, wird die MVI keine Anwendung und wird als &quot;MVI = SPM&quot; angezeigt werden. Zusätzlich, wenn die Konzentration der Zellen außerhalb des MVI Betriebsbereich, eine Botschaft der &quot;MVI = Out of Range&quot; wird angezeigt. </li><li> Um das aktuelle Histogramm speichern, berühren Sie die Fertig-Symbol. Dieser speichert die Daten mit dem Namen &quot;Test XXX&quot; wobei XXX die entsprechenden Prüfnummer. </li><li> Um manuell Tor des Histogramms, berühren, um die Kurve Fit Zählergebnisse Taste umzuschalten. Damit ist die Standard-Gating Erfassungsmodus. Dann schieben Sie die blau-Gating-Marker, die Tore gesetzt, oder tippen Sie the Pfeile links und rechts (diese erscheint nach Berühren der blau-Gating-Marker) für feine Tor Anpassungen werden nur die Zellen zwischen den Markierungen werden gezählt. Das Histogramm vertikale Skala wird automatisch basierend auf der Kurve-fit Bevölkerung Amplitude skaliert. Um diese vertikale Skala einstellen, streichen Sie den Bildschirm nach oben oder unten in der Histogramm-Region. </li><li> Berühren Sie die Gated Graf Ergebnisse, um die Anzeige zu Kurvenanpassung Modus zurückzukehren und Kurvenanpassung Modus den Standard-Erfassungsmodus. </li><li> Dann drücken Sie die Fertig-Symbol, um die Ergebnisse zu speichern und zum Startbildschirm zurück. Um die Ergebnisse zu löschen, drücken Sie auf das Symbol Löschen jederzeit dauerhaft zu löschen, die Ergebnisse des Tests. </li></ol> 4. Analyse von bereits erfassten Daten <ol><li> Um einen gespeicherten Test zu öffnen, drücken Sie die Histogramm-Symbol auf dem Home-Bildschirm. </li><li> Ikonen für bis zu neun gespeicherten Histogramme werden auf dem Bildschirm angezeigt werden.Drücken Sie auf das entsprechende Symbol für den Test von Interesse oder drücken Sie die Page Up oder Page Down Symbole, um weitere Testergebnisse anzuzeigen. </li><li> Jedes Histogramm wird entweder in der Curve Fit Count-Modus oder Gated Count-Modus geöffnet werden, je nachdem, wie es gespeichert wurde. In Gated Count-Modus können Gating Marker positioniert, indem jede Markierung blau-Gating unabhängig Wünsche offen. Auto-Tor durch Berühren Sie auf die gewünschte Spitze. Berühren Sie, um zwischen dem Grafen und Gated Curve Fit Graf Modi zu wechseln. </li><li> Drücken Sie die Zoom-und Unzoom Symbole, um die vertikale Skalierung des Histogramms einstellen. Die vertikale Skala kann auch durch vertikal einen Finger über das Display nach oben (zu erhöhen) oder unten (leiser) direkt geändert werden. </li><li> Drücken Sie die Löschen-Symbol dauerhaft zu löschen, die Ergebnisse des Tests. </li><li> Drücken Sie die Fertig-Symbol, umschließen Sie die Testergebnisse und kehren Sie zum Startbildschirm zurück. (Hinweis: Zoom-Ansicht nicht gespeichert werden.) </li></ol> 5. Übertragen von Daten an einen PC <ol><li> Daten können auf einen PC mit der Orflo Moxichart Anwendung oder über USB-Transfer, indem Sie das Moxi Z USB-Kabel an einen PC oder Mac übertragen werden. Für Bluetooth-Übertragung (MoxiChart), zuerst die Bluetooth-ID des Geräts, indem Sie die Bluetooth-Symbol auf dem Home-Bildschirm des Moxi Z-Einheit. </li><li> Auf dem Computer, öffnen Sie die MoxiChart Anwendung und klicken Sie auf das Bluetooth-Symbol in der oberen rechten Ecke. Dann wählen Sie das Gerät, das mit dem Bluetooth-ID des Moxi Z entspricht, und wählen Sie einen Zielordner für die hochgeladenen Dateien. </li><li> Moxichart wird automatisch übertragen Sie alle Dateien in das angegebene Verzeichnis über die Bluetooth-Verbindung. </li><li> Dateien per Bluetooth (MoxiChart) oder USB übertragen kann geöffnet werden und Analysedirekt mit der Anwendung MoxiChart zed oder, weil sie formatiert sind. csv-Dateien, können sie direkt in Microsoft Excel oder andere Programme Datenanalyse werden für zusätzlich / custom Analyse eröffnet. </li></ol> 6. Repräsentative Ergebnisse Um die Präzision und Genauigkeit des Moxi Z beurteilen, Grafen von HEK-293 Zellen, HeLa-Zellen, CHO-K1-, Hefe-, Jurkat E6-1-Zellen, PC12-Zellen, und Bead-Suspensionen mit Konzentrationen im Bereich von 0-500,000 Zellen präzise kalibriert / ml (Typ M Kassette) und 0-2-2.5e 6 Zellen / ml (Typ S-Kassette) wurden gezählt und verglichen mit einem Coulter Z2 und die Moxi Z. Alle Säugerzellen wurden von der American Type Culture Collection (ATCC) erhalten und kultiviert wie zuvor beschrieben, 11.07. Kurz gesagt, wurden Zellen in Suspension in 75 mm 2 Gewebekulturflaschen mit Kern Medien RPMI 1640 (Jurkat E6-1-, PC12), MEM (HEK-293-, HeLa-Zellen) oder F12K (CHO-K1-Zellen) kultiviert. Alle Medien wurden ergänzt mit10% fötalem Rinderserum und 100 U penicillin/100 ug Streptomycin. Die Flaschen wurden in einem Inkubator bei 37 ° C mit 5% CO 2 gehalten. Die Zellen wurden alle 3 Tage passagiert. Hefezellen (X5, C. albicans, Vin 13) gespendet wurden und sofort verwendet, wie zur Verfügung gestellt. Erstmuster Konzentrationen von ~ 500.000 Zellen / ml für den Typ M-Kassette Daten und ~ 2-2.5e 6 Zellen / ml für die Type S-Kassette wurden unter Verwendung des Coulter Z2. Nachfolgende theoretische Konzentrationen wurden durch serielle Verdünnungen mit physiologischer Puffer (Isoton II oder Hanks Balanced Salt Solution) vorbereitet. Bei jeder Konzentration wurden identische Proben von beiden Systemen gemessen und gegen einander. Wie in 2 ersichtlich ist, gab es eine starke lineare Korrelation (Typ M r 2&gt; 0,9886, Typ S r 2&gt; 0,9781) zwischen den Zählungen für alle Proben. Die Konzentrationen wurden auch zu erwarten / theoretische Zellkonzentrationen sowohl mit dem Moxi Z und der Z2 verglichen ( <strong&gt; Abbildung 3) und erneut gezeigt, starke lineare Korrelationen (Typ M r 2&gt; 0,9758, Typ S r 2&gt; 0,9774). Um die Genauigkeit zu bestimmen, wurden HEK-293 und HeLa-Zellzahlen unter Verwendung des Coulter Z2, Z Moxi und eines Hämocytometers. Der mittlere Variationskoeffizient (CV, n = 19) der Zellzahlen unter Verwendung des Moxi Z (Typ M-Kassette), Coulter Z2 und eine Zählkammer wurden berechnet mit Lebenslauf von 5 separate Zählungen in den 200.000-300.000 Zellen / ml-Bereich gemessen wird. Die geringe durchschnittliche CV (4) des Moxi Z ist vergleichbar mit der Z2 und steht stellvertretend für die hohe Genauigkeit nur erreichbar mit einem Coulter-basierten Zähltechnologien. Genauigkeit der Abmessungen des Moxi Z Instrument wurde als nächstes untersucht durch die Messung der erworbenen präzisionskalibriert Polystyrol-Kügelchen. Mittleren Teilchengröße Messungen (n = 5) von Moxi Z wurden gegen Hersteller berichtet Größen aufgetragen. Wie ersichtlich in Abbildung 5, die Moxi Z Messungen konsistent aufeinander abgestimmt die Hersteller Werte mit einem hohen lineare Korrelation (r 2 = 0,9989). Jenseits von Größe und Anzahl der Informationen, bietet Moxi Z ein Reagenz-freie, allgemeine Beurteilung der Säugetier-Zellkultur Gesundheit mit einem gemeldeten Moxi Viability Index (MVI) Wert. Dieser Index wird aus einem Verhältnis der Anzahl der Zellen in der Population von Interesse mit Bezug auf die gesamten Partikel zählt erzeugt. Um die MVI Messmöglichkeiten zu demonstrieren, wurden MVI Lesungen (Typ M-Kassette) auf Standard-Handbuch und durchflusszytometrische Live / Dead-Messtechnik verglichen. Die Populationen der Toten (&lt;10% lebensfähig) HEK-293-und HeLa-Zellen wurden über Nacht durch Inkubation von lebenden Zellen in einem Nährmedium-frei, Natriumfluorid enthält Verdünnungsmittel (Isoton II, Beckman Coulter) bei 37 ° C erstellt Kontrollierte theoretische Lebensfähigkeit wurden durch Mischen ratiometrisch bekannten Konzentrationen von lebenden und toten Zellen hergestellt. Res.eratung Lösungen wurden für die Lebensfähigkeit Ebenen in zweifacher Ausfertigung mit beiden Hämozytometer Zählungen und Messungen Durchflusszytometer analysiert. Hämocytometer Messungen wurden unter Verwendung eines traditionellen 50/50 Mischung von 0,1% Trypan Blau Lösung und Zellen. Durchflusszytometrie-Messungen wurden unter Verwendung eines Guave PCA-Zytometer (Merck) unter Verwendung des Viacount Reagens und die Hersteller-Protokoll spezifiziert (Merck). Wie in Abbildung 6 dargestellt, waren die Pearson-Korrelationskoeffizient Werte (r 2) &quot;der linearen Anpassung der MVI Daten zu analogen Hämozytometer Lebensfähigkeit Prozentsätze für&quot; HEK-Zellen und HeLa-Zellen r 2 = 0,9937 und r 2 = 0,9728, beziehungsweise. Diese Ergebnisse zeigen, dass dieser Ansatz Kultur Gesundheitsinformationen bietet in einem breiten Spektrum von Zellvitalitäten, die den Vergleich zu den Live / Dead-Messungen mit einem Durchflusszytometer oder Hämozytometer. Tabellen und Abbildungen (Von Dittami et al, 2011.) 15: Abbildung 1. Dünnschicht-Coulter-Zählung: Elektrischer Strom wird durch die Zelle Sensing Zone der Dünnschicht-Kassette weitergegeben. Wie Zellen einzelne Datei fließen durch die CSZ, werden die momentanen Erhöhungen der Spannung durch die Moxi Z. gemessen <img alt="2" src="/files/ftp_upload/3842/3842fig2.jpg" /> Abbildung 2. Moxi Z zählt sind vergleichbar mit Coulter Z2 zählt. Gleiche gilt der Zelle und Kügelchen Suspensionen wurden unter Verwendung sowohl eines Coulter Z2 und die Moxi Z. gemittelt Zählwerte (n = 3-4) der Moxi Z zählt wurden in Bezug auf die gezeichneten Coulter Z2 entsprechenden Zählungen und R 2-Werte der linearen Anpassung bestimmt. A) Typ M-Kassette - Konzentrationsbereich von 0 - 500.000 Zellen / ml. B) Typ S-Kassette - Konzentrationsbereich von 0 - 2-2.5e 6 / ml.Error Balken zeigen ± 1 Standardabweichung. Abbildung 3. Moxi Z Konzentrationsmessungen vs theoretischen Zellkonzentrationen. A) Typ M-Kassette - Theoretische Konzentration Werte wurden aus Verdünnungsreihen von zunächst 500.000 Zellen / ml Probe vorbereitet B) Typ S-Kassette -. 2.5e 6 Zellen / ml Probe - Theoretische Konzentration Werte von Verdünnungsreihen eines anfänglichen 2 hergestellt. Fehlerbalken zeigen ± 1 Standardabweichung. <img alt="Abbildung 4" src="/files/ftp_upload/3842/3842fig4.jpg" /> Abbildung 4. Variationskoeffizient für die Coulter Z2, Moxi Z (Typ M-Kassette) und Hämocytometer. Der mittlere Variationskoeffizient (CV, n = 19) von HEK-293-und HeLa-Zellzahlen für jeden Ansatz wurden mit Lebensläufen aus 5 separaten gemessen berechnet Grafen von HEK-293-und HeLa-Zellen in der 200.000 - 300.000 Zellen / ml-Bereich. Fehlerbalken zeigen ± 1 Standardabweichung. Abbildung 5. Die Genauigkeit der Messungen mit Partikelgröße Moxi Z-Moxi Z gemessen Perlgröße (Typ M-Kassette) vs Hersteller berichtet Perlgröße. Fehlerbalken zeigen ± 1 Standardabweichung. <img alt="6" src="/files/ftp_upload/3842/3842fig6.jpg" /> Abbildung 6. Kultur Health Assessment mit MVI - Vergleich von MVI-Messungen (Typ M-Kassette) und Guava PCA Viacount Rentabilität im Vergleich zu Visual, Trypan-Blau gefärbten Lebensfähigkeit zählt mit einer Zählkammer.

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORFLO+Moxi+Z:+Revolutionizing+electronic+cell+count+accuracy+and+cell+viability+estimates+through+curve+fitting.">ORFLO Moxi Z: Revolutionizing electronic cell count accuracy and cell viability estimates through curve fitting.</a> Life Science Magazine, VWR International. , (2011).</li></ol>

Gregory M. Dittami und HE Ayliffe sind Angestellte der Orflo Technologies, dem Unternehmen, entwickelt, fertigt und vertreibt das Moxi Z. Richard D. Rabbitt bietet Teilzeit, bezahlt Beratungsexpertise zur Orflo Technologies. Produktion und freien Zugang zu diesem Artikel wird von Orflo Technologies gesponsert.

Die zugrunde liegende Implementierung der Coulter-Ansatz kann extrem deterministische der Genauigkeit der Messungen. Ein kritischer Bereich ist die Korrektur der Roh-Zellzahlen für &quot;Zufall Ereignisse&quot; wo zwei oder mehrere Zellen gleichzeitig passieren durch die Öffnung und werden als ein einziges elektrisches Ereignis gemessen. &quot;Der Zufall Ereignisse&quot; dazu beitragen, Fehler, die auf einer Reihe von Faktoren wie Größe der Zellen und Grad der Clusterbildung (Davis et al 1967) 6 variiert. Als ein Ergebnis kann die erforderliche Korrektur für zufällig jeder gegebenen Probe nicht vorhergesagt werden, variiert stark zwischen den Zellen / Partikel-Typen und sogar zwischen verschiedenen Kulturen identische Zelltypen. Etablierte Theorie, erste Veröffentlichung von Coulter verankert, beinhaltet das Aufbringen einer logarithmischen Anpassung der rohen setzt auf der Anzahl der Ereignisse beobachtet und einer empirisch bestimmten, Partikel-Korrekturfaktor, Z. Während die genaue Zählungen kann richtig mit t erreicht werdenseine Korrektur Algorithmus wird in der praktischen Anwendbarkeit aufgrund der großen Variationen in z-Werte zwischen verschiedenen Zelltypen und der damit verbundenen Schwierigkeit genaue Vorhersage der Wert begrenzt. Hier, mit dem &quot;echten&quot; Zellzahl von der Moxi Z Kurvenanpassung in Verbindung mit dem Zusammentreffen Korrekturalgorithmus identifiziert, die Moxi Z dynamisch korrigiert Zufall auf die tatsächliche Konzentration zu erreichen. Wenn auf Ergebnisse, die mit High-End-Coulter Z2 verglichen produzierte das Moxi Z vergleichbar zählt in einem Konzentrationsbereich von 0 - 500.000 Zellen / ml (Typ M-Kassette) und 3.000, - 2-2.5e 6 Zellen / ml (Typ S-Kassette) . Darüber hinaus erzielt die Moxi Z diese Zahl mit einer Genauigkeit ähnlich niedrige Variationskoeffizienten zählt und in einer Präzision im Partikelmessung, die charakteristisch für den Goldstandard, Coulter-Technik in Zellzählung ist. Darüber hinaus deuten die hier präsentierten Daten, dass die Moxi Z können wertvolle Informationen liefernin Bezug auf die allgemeine Gesundheit der Zellkulturen. Das Moxi Z nutzt die Kurvenanpassung Ansatz mit einer proprietären Software-Algorithmus, um diese Kultur des Gesundheitszustandes festzustellen. Morphologische Veränderungen mit dem Zelltod assoziiert, wie blebbing, auseinander zu brechen, und die Volumenproduktivität Verzerrungen (Kataoka und Tsuruo 1996 Liegler et al 1995, Sheridan et al 1981) 12-14, machen es möglich, die Moxi-Z zu impedimetrischen Unterschiede zwischen unterscheiden leben Populationen und tote Zelle / Schutt Populationen. Die MVI wird durch die Analyse der Kultur / Partikelgrößenverteilung auf die relativen Beiträge der Partikel-Ablagerungen, abgestorbene Zellen geschrumpft, und die Kurve ausgestattete Zellpopulation von Interesse zu identifizieren generiert. Der MVI-Wert spiegelt damit eine Bevölkerung Index oder ratiometrisch Maßnahme der monodispersen Population zählt in Bezug auf das gesamte Teilchen Bevölkerung Profil. Dieser Wert ist dann algorithmisch-based angepasst Bevölkerungsstatistik und empirische Beobachtungen. Because der MVI schaut auf andere Parameter als traditionelle Ansätze Färbung, ist nicht zu erwarten, diese Techniken zu spiegeln, sondern bietet eine wertvolle Alternative Blick in die Gesundheit einer Zellkultur, insbesondere im Hinblick auf den Trümmern und mikrobiellen Verunreinigungen. Zusammenfassend ist die Moxi-Z Zellzahl-Messgerät ein ultra kleines Tischgerät, das eine präzise und reproduzierbare Tests von Zellzahl, Zellgröße und Gesundheit Zelle mit dem Coulter-Prinzip und Kurvenanpassung Software-Algorithmen mit einer patentierten Dünnschicht-Sensor-Technologie kombiniert wird, liefert . Mit dem Typ M-Kassette ist in der Lage Zählen von Partikeln im Bereich von 4 bis 25 Mikrometer im Durchmesser (dynamischer Bereich von 2 - 34 Mikrometer), in 8 Sekunden. Mit dem Typ S-Kassette ist in der Lage Zählen von Partikeln im Bereich von 3 bis 20 Mikrometer im Durchmesser (dynamischer Bereich von 2 - 26 Mikrometer), in 15 Sekunden. Außerdem führt die Moxi-Z Gesundheitszustandes ohne die Verwendung von Reagenzien. Es ist viel weniger Arbeit intensive und subjektiver als manuelles Zählen. Im Vergleich zu Standard-Coulter-Zählung, die Moxi schneller ist, deutlich kleiner, bietet mehr Informationen, benötigt deutlich weniger Wartung ist einfacher zu bedienen, und ist ein Bruchteil der Kosten.

<table border="1" ><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> ORFLO Diluent </td><td> ORFLO </td><td> MXA006 </td><td></td></tr><tr><td> Kassetten-Dispenser </td><td> ORFLO </td><td></td><td> Speichert bis zu 25 Kassetten für bequeme Vorratsbehälter </td></tr><tr><td> USB-Kabel </td><td> ORFLO </td><td></td><td> Verbindungsleitung Messgerät PC / Mac oder Netzteil </td></tr><tr><td> Power Adapter (US-und EU-Modelle) </td><td> ORFLO </td><td></td><td> Verbindet USB-Kabel an eine Steckdose </td></tr><tr><td> USB Flash Drive </td><td> ORFLO </td><td></td><td> Mox Storesi Z-Software und Benutzerhandbuch </td></tr><tr><td> Calibration Check Perlen </td><td> ORFLO </td><td></td><td></td></tr><tr><td> Elektronische Kalibrierung Cassette </td><td> ORFLO </td><td></td><td> Elektronische Kassette für die Überprüfung der ordnungsgemäßen Betrieb des Systems und Kalibrierung </td></tr></tbody></table>

determination of mammalian cell counts, cell size and cell health using the moxi z mini automated cell counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

Watch this Scientific Journal Video about Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter at JoVE.com

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Das Moxi Z Miniatur automatisierte Zelle Zähler ist ein neuartiges Instrument, das die Coulter-Prinzip kombiniert mit der patentierten Dünnschicht-Sensor-Technologie und einer proprietären Software-Algorithmus, um Sizing durchführen und das Zählen von einem breiten Größenbereich der Partikel sowie auf die allgemeine Gesundheit der monodispersen bestimmen Säugerzellkulturen. Dieses Protokoll beschreibt die Verwendung dieses Instruments zum Zählen und Überwachung des Gesundheitszustands der Zellkulturen.

Bestimmung der Säugetier-Zellzahlen, Größe der Zellen und Zell Gesundheit Mit dem Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

determination-mammalian-cell-counts-cell-size-cell-health-using-moxi

Research

JoVE Journal

Biology

24.6K Aufrufe.  Orflo Technologies. Das Moxi Z Miniatur automatisierte Zelle Zähler ist ein neuartiges Instrument, das die Coulter-Prinzip kombiniert mit der patentierten Dünnschicht-Sensor-Technologie und einer proprietären Software-Algorithmus, um Sizing durchführen und das Zählen von einem breiten Größenbereich der Partikel sowie auf die allgemeine Gesundheit der monodispersen bestimmen Säugerzellkulturen. Dieses Protokoll beschreibt die Verwendung dieses Instruments zum Zählen und Überwachung ...

Serie: Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Langfristige Imaging Säugetierzellen mit Wide-Field-Mikroskopie

Forschung

Biologie

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulse-Chase-Analyse von N-verknüpften Zuckerketten von Glykoproteinen in Säugerzellen

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

MISSION esiRNA für RNAi-Screening in Säugerzellen

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Bestimmen Zellzahl während der Zellkultur mit dem Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse des Zellzyklus Position in Säugerzellen

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Die Messung der Hämsynthese Levels in Säugerzellen

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Erzeugung von Plasmid-Vektoren, die FLAG-markierten Proteine ​​Nach der Verordnung der Menschen Elongation Factor-1α Promoter Mit Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Direkter Protein Lieferung Säugerzellen mit Zellpermeable Cys<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt; Zink-Finger-Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Anaerobe Wachstum und Erhalt von Säugetieren Zelllinien

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...