1. Instrument Setup <ol><li> Schalten Sie den Spektrofluorometer und warmlaufen lassen für 1 Stunde. </li><li> Mit Excel erstellen Sie eine Datei mit den Standards und Sample-Daten, wie in Abbildung 1 gezeigt und speichern Sie sie in Comma Separated Values ​​&quot;csv&quot;-Format. <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Standards und Proben Daten. </li><li> Set up Fluorometer wie folgt: <ol><li> Klicken Sie auf die <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="Icon 1" /> Taste, wird diese öffnen Sie die Analysemethode Fenster in Abb. 2 dargestellt. <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Analysemethode Fenster, Registerkarte Allgemein. Machen Sie die folgende Auswahl: <ul><li> In der &quot;Measurement&quot;-Feld, wählen Sie Photometrie aus dem Pulldown-Menü. </li><li> In &quot;Operator&quot;-Feld, geben Sie die entsprechenden Betreiber zu nennen. </li><li> Es besteht keine Notwendigkeit, Informationen in der &quot;Instrument&quot; Feld eingeben. Dies erfolgt automatisch durch die Software selektiert. </li><li> Das &quot;Sampling&quot;-Feld ist inaktiv, wenn mit der Mikroplatten-Reader. </li><li> Geben Sie Kommentare, die Sie in dem Bericht in der &quot;Kommentar&quot;-Feld enthalten sein soll. </li><li> In der &quot;Accessory&quot;-Feld, geben Sie Informationen im Zusammenhang mit dem Zubehör für diesen Test verwendet. </li><li> Beachten Sie die Schaltflächen auf der rechten Seite des Fensters. </li><li> Der &quot;Load&quot;-Taste erlaubt das Laden zuvor gespeicherte Analyse-Methoden. </li><li> Die Schaltfläche &quot;Speichern&quot; ermöglicht die Aktualisierung eine Reihe von Test-Parameter oder Analysemethode zuvor gespeichert und geladen, mit dem gleichen Namen. </li><li> Die Taste ermöglicht das Speichern einer neuen Reihe von Test-Parameter oder Analysemethode unter einem gewünschten Namen &quot;Save As&quot;. </li></ul> Jetzt auf dem &quot;Quantifizierung&quot; Registerkarte klicken, siehe Abbildung 3 und die folgende Auswahl. <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Analysemethode Window, Quantifizierung Registerkarte. <ul><li> In der &quot;Quantifizierung Typ&quot;-Feld, wählen Wellenlänge. Dies zeigt die Fluoreszenz Lesen gemacht wird mit der gewählten Wellenlänge sein. </li><li> In der &quot;Calibration Type&quot;-Feld, wählen Sie 1. Ordnung. </li><li> In der &quot;Anzahl der Wellenlängen&quot;-Feld, wählen Sie 1. </li><li> In der &quot;Konzentration Einheit&quot; Feld ng / mL. </li><li> Lassen Sie unselected die Felder für &quot;Manuelle Kalibrierung&quot; und &quot;Force Kurve durch Null&quot;. </li><li> In der &quot;Nachkommastelle&quot;-Feld, wählen Sie 2. </li><li> In der &quot;Lower Grenzkonzentration&quot;-Feld, 0 eingeben. </li><li> In der &quot;Upper Konzentrationsgrenze Feld 10000. </li></ul> Jetzt auf dem &quot;Instrument&quot; Registerkarte klicken und die folgende Auswahl. <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Analysemethode Fenster Instrument tab <ul><li> In der &quot;Data-Modus:&quot; Wählen Sie im Feld Fluoreszenz. </li><li> Die &quot;Chopping speed&quot;-Feld ist inaktiv zu diesem Zeitpunkt, es nur für Phosphoreszenz Messungen verwendet. </li><li> In der &quot;Wavelength-Modus&quot;-Feld, wählen Sie Beide WL fixiert. </li><li> In der &quot;EX / WL1&quot; Feld 480nm. </li><li> In der &quot;EM / WL1&quot; Feld 520nm. </li><li> Alle anderen Felder unter EX und EM sind in dieser Zeit inaktiv. </li><li> In der &quot;EX Schlitz&quot;-Feld wählen 5.0nm aus dem Pulldown-Menü. </li><li> In der &quot;EM Schlitz&quot;-Feld wählen 5.0nm aus dem Pulldown-Menü. </li><li> Lassen Sie unselected das Feld vor der &quot;PMT Voltage 1-1000V&quot;-Feld. </li><li> In der &quot;PMT Voltage&quot;-Feld, wählen Sie 700V aus dem Pulldown-Menü. </li><li> In der &quot;Auto-Statistik calc. Number&quot;-Feld wählen Sie 2. </li><li> In der &quot;Repliziert&quot; Feld auswählen 1. </li><li> In der &quot;Integration time&quot; Feld auszuwählen 0.1s. &lt;/ Li&gt; <li> In der &quot;Delay&quot;-Feld eingeben oder 0s. </li></ul> Klicken Sie auf die Registerkarte &quot;Monitor&quot;. <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Analysemethode Fenster die Registerkarte Monitor. <ul><li> In der &quot;Y-Achse&quot;, &quot;Max:&quot;-Feld, wählen Sie 10000 </li><li> In der &quot;Y-Achse&quot;, &quot;Min:&quot;-Feld, wählen 0. </li><li> Wählen Sie das Feld links neben der &quot;Open Datenverarbeitung nach Datenerfassung&quot;. </li><li> Lassen Sie unselected der Box auf der linken Seite des &quot;Bericht drucken nach Datenerfassung&quot;, wenn Sie einen Bericht automatisch gedruckt werden, nachdem die Lesungen abgeschlossen werden soll. </li></ul> Klicken Sie auf den &quot;Report&quot;-Registerkarte, siehe Abbildung 6. <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="Abbildung 6" /> Abbildung 6. Analysemethode Fenster, Report Tab. <ul><li> In den &quot;Output&quot;-Feld, wählen Sie Bericht drucken aus dem Pulldown-Menü. Sie können auch wählen Sie &quot;Verwenden Sie Microsoft Excel&quot;, wenn Sie die Daten nach Excel exportiert oder &quot;Use Print Generator Sheet&quot; bei Verwendung des optionalen add on Programm Report Generator, die es generiert maßgeschneiderte Berichte werden wollen. </li><li> Klicken Sie die Kontrollkästchen für die Elemente, die Sie in den Bericht aufgenommen werden. </li><li> So speichern Sie alle ausgewählten Parameter in den verschiedenen Tabs des &quot;Analysis Method&quot;-Fenster, klicken Sie erneut auf die Registerkarte &quot;Allgemein&quot; und speichern Sie sie unter einem Dateinamen und in einem Ort, wo Sie sie für zukünftige Verwendung finden konnte, und klicken Sie auf &quot; OK &quot;-Taste, um das Fenster zu schließen. </li><li> Damit ist die Inbetriebnahme des Instrumentes Prüfparameter. </li><li> Jetzt richten wir Ihnen die Mikroplatten-Reader. </li><li> Klicken Sie auf die <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="Icon 2" /> Button, der die MPR-Setup-Fenster auf der Oberseite bringt. </li></ul> Wählen Sie Vertiefungen für die Standards und die Proben in der MPR-Setup-Fenster / Plate Register, wie in Abbildung 7 dargestellt. <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="Abbildung 7" /> Abbildung 7. MPR-Setup-Fenster, Plate Registerkarte. <ul><li> Klicken Sie auf die A1 Position und ziehen Sie die Maus auf E1, dann klicken Sie auf den <img src="/files/ftp_upload/2465/2465icon3.jpg" alt="Icon 3" /> -Taste. Diese wählt die Positionen für die Standards verwendet werden. Diese Positionen werden in grüner Farbe hervorgehoben werden. </li><li> Jetzt müssen wir Positionen F1 bis H3 wählen für die Proben. Klicken und Ziehen mit der Maus ab Position F1, um H3 Position und klicken Sie dann auf die <img src="/files/ftp_upload/2465/2465icon4.jpg" alt="Icon 4" /> drücken, wird dieses Highlight dieser Positionen in gelber Farbe. </li><li> Dann wiederholen Sie den gleichen Vorgang für die Positionen A2 bis E3, um die Positionen für die Probe-Messungen zu wählen. </li><li> Jetzt wollen wir laden Sie die Datei Comma Separated Variable (csv-Datei) im Voraus mit den Standards und Probe bereitgestellten Informationen. </li></ul> Klicken Sie auf das &quot;Well Informationen&quot; auf der Registerkarte &quot;MPR Setup-Fenster&quot;, wie in Abbildung 8 dargestellt. <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="Abbildung 8" /> Abbildung 8. MPR-Setup-Fenster, Well Registerkarte Informationen. <ul><li> Dann klicken Sie auf den <img src="/files/ftp_upload/2465/2465icon5.jpg" alt="Icon 5" /> -Taste. Ein Windows-Explorer-Fenster öffnet sich, die wie in Abbildung 9, die es erlauben Auffinden der &quot;Well information.csv&quot;-Datei und laden es wird gezeigt. <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="Abbildung 9" /> Abbildung 9. Lädt auch Probeninformationen. Nach dem Laden der &quot;Well information&quot;-Datei, wird der &quot;Well Informationen&quot; so aussehen in Abbildung 10 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="Abbildung 10" /> Abbildung 10. Loaded Probe auch Informationen. </li></ul></li></ol></li></ol> 2. PicoGreen Assay Probenvorbereitung <ol><li> Bereiten 1X TE-Puffer (10 mM Tris-HCl, 1 mM EDTA) durch Zugabe von 1 ml 20 X TE-Pufferbis 19 mL DI H 2 O. </li><li> Bereiten PicoGreen Reagenzlösung durch Zugabe von 50 ul 200 X PicoGreen Reagenz auf 9,95 ml 1 x TE-Puffer. </li><li> Bereiten Standards (Lambda dsDNA), indem serielle Verdünnungen von Lager dsDNA (100 ug / mL). Bereiten Sie zuerst eine Lösung von 50 ug / ml dsDNA durch Zugabe von 25 ul Lager dsDNA (100 ug / ml) und 25 ul 1 x TE-Puffer. Anschließend werden die Standard-Lösungen, indem Sie die angegebenen Mengen an dsDNA und Puffer wie in der Tabelle unten aufgelistet: <table border="1"><tbody><tr><td> Die Konzentration der dsDNA </td><td> Volumen der dsDNA </td><td> Volumen Puffer </td></tr><tr><td> 1 pg / mL </td><td> 20 ul von 50 ug / mL dsDNA </td><td> 980 ul 1 X TE-Puffer </td></tr><tr><td> 0,1 ug / mL </td><td> 100 ul von 1 pg / mL dsDNA </td><td> 900 ul 1 X TE-Puffer </td></tr><tr><td> 0,01 g / mL </td><td> 100 ul von 0,1 ug / ml dsDNA </td><td> 900 ul 1 X TE-Puffer </td></tr><tr><td> 0,001 g / mL </td><td> 100 ul von 0,01 ug / ml dsDNA </td><td> 900 ul 1 X TE-Puffer </td></tr></tbody></table></li></ol> 3. Measurement of Standards and Probenfluoreszenz <ol><li> Je 150 ul von jedem Standard in die Vertiefungen A1-E1 einer 96-Well-Platte, pro Bild 7, und nach der folgenden Tabelle: <table border="1"><tbody><tr><td> Gut </td><td> Standard </td></tr><tr><td> A1 </td><td> 1 pg / mL </td></tr><tr><td> B1 </td><td> 0,1 ug / mL </td></tr><tr><td> C1 </td><td> 0,01 g / mL </td></tr><tr><td> D1 </td><td> 0,001 g / mL </td></tr><tr><td> E1 </td><td> 1 X TE-Puffer </td></tr></tbody></table></li><li> Je 150 ul unbekannten Proben in die Vertiefungen F1 bis H3, in Abbildung 7 </li><li> Gießen PicoGreen Lösung in Schritt 1.2 in einen Trog für Mehrkanal-Pipette Einsatz konzipiert vorbereitet. Verwenden Sie eine Mehrkanal-Pipette auf 150 ul PicoGreen Lösung in die Vertiefungen A1-H3 zu liefern. Die Lösung und Standards vorsichtig mit Pipette. </li><li> Legen Sie Platte in dunklen Bereich und warten 2-5 Minuten für die Reaktion zu entwickeln. </li></ol> 4. Repräsentative Ergebnisse Eine gute Kalibrierkurve wird unter Verwendung der verschiedenen Parameter, die von der F-7000-Software bei der Messung der Standards und die Berechnung der Kalibrierkurve von der Software zur Verfügung gestellt. Das Bestimmtheitsmaß gibt die Güte der Anpassung der gemessenen Standards und die berechnete Eichkurve. Je näher dieser Wert auf &quot;1&quot;, der besser ist die Anpassung des Messwertes und Eichkurve. Wenn der Wert ist weit von &quot;1&quot;, müssen die Standards neu vermessen werden, vorbereitet erneut, oder die Tauglichkeit der Kalibrierkurve geändert. Abbildung 11 zeigt ein Beispiel für eine Eichkurve mit einer berechneten Bestimmtheitsmaß = 0,99993, für die Quantifizierung von dsDNA mit PicoGreen Farbstoff erhalten. <img src="/files/ftp_upload/2465/2465fig11.jpg" alt="Abbildung 11" /> Abbildung 11. Beispiel für dsDNA Kalibrierkurve.

<ol>
	<li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation.">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Analytical Biochemistry. 249 (2), 228-238 (1997).</li></ol>

Luis A. Moreno und Kendra L. Cox von Hitachi High Technologies America, dass das Instrument in diesem Artikel verwendet produziert beschäftigt.

Sorgfältige Pipettieren während der Probenvorbereitung sowie über eine stabile und empfindliche Fluoreszenz-Spektralphotometer ist unerlässlich für den Erhalt von guten und reproduzierbaren Ergebnissen. Im Falle der Fluoreszenz-Spektralphotometer für diesen Test verwendet wird vorgeschlagen mit 300 ul Endprobe Volumen im Mikroplatten-Reader. Niedrigere Volumen verringert sich die Empfindlichkeit und ein größeres Volumen kann Kreuzkontaminationen zwischen den Wells zu verursachen.

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
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<tr>
<td>Distilled H2O</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Quant-iT Picogreen dsDNA Assay Kit from Invitrogen</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>P7589</td>
<td>&nbsp;</td>
<tr>
<td>Nalge Nunc Fluorescence Microplates p/n 237108</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>237108</td>
<td>&nbsp;</td>
<tr>
<td>12 Channel Eppendorf Micropipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>3516677</td>
<td>&nbsp;</td>
<tr>
<td>1000 &mu;L Eppendorf Pipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>200 &mu;L Eppendorf Pipette </td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10 mL serological pipet</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Aluminum foil</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Pipette tips</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J1-0003</td>
<td>&nbsp;</td>
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<td>Microplate Reader Accessory for F-7000</td>
<td>Equipment</td>
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quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

Quantifizierung von DNA, vor allem in kleinen Konzentrationen, ist eine wichtige Aufgabe mit einer Vielzahl von biologischen Anwendungen, einschließlich Standard-Molekularbiologie-Assays wie Synthese und Aufreinigung von DNA-, Diagnose-Anwendungen wie die Quantifizierung der DNA-Amplifikation Produkte und Detektion von DNA-Molekülen in der Medikamentenentwicklung Vorbereitungen. Während dieses Video zeigen wir Ihnen die Leistungsfähigkeit der Hitachi F-7000 Fluoreszenz-Spektralphotometer mit einer Micro-Plate Reader Zubehör zum dsDNA Quantifizierung mittels Molecular Probes Quant-it PicoGreen Farbstoff Reagenzienkit durchführen ausgestattet. Der F-7000 Fluoreszenzspektrophotometer bietet hohe Empfindlichkeit und hohe Geschwindigkeit Messungen. Es ist ein sehr flexibles System für die Messung von Fluoreszenz, Lumineszenz und Phosphoreszenz. Mehrere Betriebsarten zur Verfügung, einschließlich Wellenlänge zu scannen, zu scannen, Photometrie und 3-D-Scan-Messung. Das Spektralphotometer ist die Empfindlichkeit im Bereich von 50 pmol Fluorescein bei der Verwendung eines 300 &amp;mgr; Probenvolumen in der Mikrotiterplatte und ist in der Lage Scan-Geschwindigkeiten von 60.000 nm / min. Es hat auch einen breiten dynamischen Bereich von bis zu 5 Größenordnungen, die für die Verwendung von Kalibrierkurven über einen weiten Bereich von Konzentrationen erlaubt. Das optische System nutzt alle reflektive Optiken für maximale Energie und Sensibilität. Die Standard-Wellenlängenbereich von 200 bis 750 nm, und kann bis 900 nm erweitert werden, wenn mit einem der optionalen Nah-Infrarot-Photomultiplier. Das System ermöglicht optional Temperaturregelung für die Platten-Lesegerät 5 bis 60 Grad Celsius mit einem optionalen externen temperierten Flüssigkeit Thermostaten. Die Mikroplatten-Reader ermöglicht den Einsatz von 96-Well Mikrotiterplatten, und die Messung der Geschwindigkeit für 96 Vertiefungen von weniger als 60 Sekunden bei Verwendung der Kinetik-Modus. Software steuert für die F-7000 und Mikroplatten-Reader sind auch sehr flexibel. Die Proben können entweder in Spalte oder Zeile Formate eingestellt werden und eine beliebige Kombination von Brunnen kann für die Probe-Messungen gewählt werden. Dies ermöglicht eine optimale Ausnutzung der Mikroplatten. Darüber hinaus ermöglicht die Software den Import Mikrotiterplatten Beispielkonfigurationen in Excel erstellt und gespeichert in Comma Separated Values ​​oder &quot;csv&quot;-Format. Microplate Mess-Konfigurationen können gespeichert und wieder aufgerufen werden, indem die Software für Komfort und erhöhte Produktivität. Daten Ergebnisse kann auf einem Standard-Bericht, Excel, oder einem optionalen Report Generator Program.

Watch this Scientific Journal Video about Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye at JoVE.com

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Demonstration der Quantifizierung von dsDNA mit Molecular Probes PicoGreen Farbstoff und Hitachi F-7000 Fluoreszenz-Spektralphotometer mit einem Mikroplatten-Reader Zubehör ausgestattet.

Quantifizierung von dsDNA mit dem Hitachi F-7000 Fluoreszenz-Spektralphotometer und PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

quantification-dsdna-using-hitachi-f-7000-fluorescence

Research

JoVE Journal

Biology

24.4K Aufrufe.  Hitachi High Technologies America. Demonstration der Quantifizierung von dsDNA mit Molecular Probes PicoGreen Farbstoff und Hitachi F-7000 Fluoreszenz-Spektralphotometer mit einem Mikroplatten-Reader Zubehör ausgestattet.

Serie: Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Mikrovolumen Proteinkonzentrationsbestimmung mit dem NanoDrop 2000c Spektrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Forschung

Biologie

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zeitraffer-Imaging der Mitose Nach siRNA-Transfektion

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Konzentrationsbestimmung von Nukleinsäuren und Proteinen Mit dem Micro-Volumen Bio-spec Nano-Spektralphotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absolute Quantenausbeute Messung von Pulverproben

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Visualisierung von Caenorhabditis elegans Cuticle Strukturen mit der lipophilen Vital Dye, DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Beobachtung und Quantifizierung der Telomere und repetitiver Sequenzen mittels Fluoreszenz<em&gt; In Situ</em&gt; Die Hybridisierung (FISH) mit PNA-Sonden in<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Visualisierung und Quantifizierung von braunem und beigem Fettgewebe in Mäusen mittels [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Quantifizierung der interbakteriellen Konkurrenz mittels Einzelzell-Fluoreszenzbildgebung

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...