1. Strumento Setup <ol><li> Accendere il spettrofluorimetro e lasciare scaldare per 1 ora. </li><li> Utilizzo di Excel crea un file con gli standard ed i dati del campione, come mostrato nella Figura 1 e salvarlo in valori separati da virgola formato &quot;csv&quot;. <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="Figura 1" /> Figura 1. Standard e campioni di dati. </li><li> Istituito fluorimetro come segue: <ol><li> Clicca sul <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="Icona 1" /> pulsante, si aprirà la finestra metodo di analisi, mostrata in Fig. 2. <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="Figura 2" /> Figura 2. Metodo di analisi finestra, nella scheda Generale. Effettuare le seguenti selezioni: <ul><li> Nel campo &quot;Misura&quot;, selezionare Fotometria dal menu a discesa. </li><li> Nel campo &quot;Operatore&quot;, inserire il nome dell&#39;operatore appropriato. </li><li> Non è necessario inserire le informazioni nel campo &quot;strumento&quot;. Questo viene selezionato automaticamente dal software. </li><li> Il campo &quot;Sampling&quot; non è attivo quando si utilizza il lettore per micropiastre. </li><li> Inserire eventuali commenti che è possibile includere nel rapporto nel campo &quot;Commenti&quot;. </li><li> Nel campo &quot;accessori&quot;, inserire le informazioni relative agli accessori utilizzati per questo test. </li><li> Nota i pulsanti sul lato destro della finestra. </li><li> Il pulsante &quot;Carica&quot; ​​permette di caricare Metodi di analisi precedentemente salvato. </li><li> Il pulsante &quot;Salva&quot; consente di aggiornare una serie di parametri di prova o di metodo di analisi precedentemente salvato e caricato, utilizzando lo stesso nome. </li><li> &quot;Salva con nome&quot; pulsante permette di salvare una nuova serie di parametri di prova o di metodo di analisi sotto il nome desiderato. </li></ul> Ora cliccate sulla scheda &quot;quantificazione&quot;, vedi figura 3 e effettuare le seguenti selezioni. <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="Figura 3" /> Figura 3. Metodo di analisi finestra, scheda quantificazione. <ul><li> Nel &quot;tipo di quantificazione&quot;, selezionare lunghezza d&#39;onda. Questo indica che la lettura della fluorescenza sarà effettuata utilizzando la lunghezza d&#39;onda selezionata. </li><li> Nel &quot;tipo di calibrazione&quot; di campo, selezionare 1 ° ordine. </li><li> Nel campo &quot;Numero di lunghezze d&#39;onda&quot;, selezionare 1. </li><li> Nella &quot;unità di concentrazione&quot;, immettere ng / ml. </li><li> Lasciare le caselle non selezionate per &quot;Calibrazione manuale&quot; e &quot;curva di Forza per lo zero&quot;. </li><li> Nella &quot;Digit dopo punto decimale&quot;, selezionare 2. </li><li> Nel &quot;basso limite di concentrazione&quot;, immettere 0. </li><li> Nel &quot;campo superiore limite di concentrazione, immettere 10000. </li></ul> Ora cliccate sulla scheda &quot;Strumenti&quot; e effettuare le seguenti selezioni. <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="Figura 4" /> Figura 4. Metodo di analisi finestra, nella scheda strumento <ul><li> Nella modalità &quot;Dati:&quot;, selezionare fluorescenza. </li><li> La &quot;velocità Tagliere&quot; campo non è attivo in questo momento, viene utilizzato solo per le misure fosforescenza. </li><li> Nella modalità &quot;Wavelength&quot;, selezionare Entrambi fisso WL. </li><li> Nel campo &quot;EX / WL1&quot; entrare 480nm. </li><li> Nella &quot;EM / WL1&quot; inserire 520nm. </li><li> Tutti gli altri campi sotto EX e EM non sono attive in questo momento. </li><li> Nella &quot;EX fessura&quot; campo 5.0nm selezionare dal menu a discesa. </li><li> Nella &quot;EM fessura&quot; campo 5.0nm selezionare dal menu a discesa. </li><li> Lasciate selezionata la casella di fronte al &quot;Tensione PMT 1-1000V campo&quot;. </li><li> Nella &quot;PMT tensione&quot;, selezionare 700V dal menu a discesa. </li><li> Nella &quot;calc Auto statistica. Numero&quot; campo selezionare 2. </li><li> Nel campo &quot;Replica&quot;, selezionare 1. </li><li> Nel &quot;tempo di integrazione&quot; campo selezionare 0.1s. &lt;/ Li&gt; <li> Nel campo &quot;Ritardo&quot; immettere o selezionare 0. </li></ul> Fare clic sulla scheda &quot;Monitor&quot;. <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="Figura 5" /> Figura 5. Metodo di analisi finestra, scheda Monitor. <ul><li> Nella &quot;asse Y&quot;, &quot;Max:&quot;, selezionare 10000 </li><li> Nella &quot;asse Y&quot;, &quot;Min:&quot;, selezionare 0. </li><li> Selezionare la casella a sinistra del &quot;Aprire il trattamento dei dati dopo l&#39;acquisizione dei dati&quot;. </li><li> Lasciate selezionata la casella a sinistra del &quot;rapporto di stampa dopo l&#39;acquisizione dei dati&quot;, a meno che non si desidera un rapporto da stampare automaticamente dopo le letture sono state completate. </li></ul> Fare clic sulla scheda &quot;Report&quot;, vedi Figura 6. <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="Figura 6" /> Figura 6. Metodo di analisi finestra, scheda Report. <ul><li> Nel campo &quot;Uscita&quot;, selezionare Stampa rapporto dal menu a discesa. Si può anche selezionare &quot;Utilizza Microsoft Excel&quot; se si desidera che i dati esportati in Excel, o &quot;Stampa Foglio Usa Generator&quot; se si utilizza il componente aggiuntivo opzionale Generator programma rapporto che permetterà generare report personalizzati fatto. </li><li> Selezionare le caselle per gli elementi che si desidera includere nel rapporto. </li><li> Per salvare tutti i parametri selezionati nelle diverse schede del &quot;metodo di analisi&quot; finestra, clicca di nuovo sulla scheda &quot;Generale&quot; e salvare con un nome di file e in una posizione in cui si riusciva a trovarli per un uso futuro, e fare clic sul &quot; OK &quot;per chiudere questa finestra. </li><li> Questo completa l&#39;impostazione dei parametri dello strumento di prova. </li><li> Ora ci sarà istituito il lettore per micropiastre. </li><li> Clicca sul <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="Icon 2" /> pulsante, che porterà la finestra Setup MPR in cima. </li></ul> Seleziona pozzi per la standard e dei campioni nel Setup MPR Finestra / Plate scheda, come mostrato nella Figura 7. <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="Figura 7" /> Figura 7. MPR finestra di installazione, scheda Plate. <ul><li> Clicca sulla posizione A1 e trascinare il mouse per E1, quindi fare clic sul <img src="/files/ftp_upload/2465/2465icon3.jpg" alt="Icona 3" /> pulsante. In questo modo selezionare le posizioni da utilizzare per gli standard. Quelle posizioni verranno evidenziati in colore verde. </li><li> Ora abbiamo bisogno di selezionare F1 posizioni H3 per i campioni. Fare clic e trascinare il mouse a partire F1 posizione, alla posizione H3 e quindi fare clic sul <img src="/files/ftp_upload/2465/2465icon4.jpg" alt="Icona 4" /> pulsante, questo metterà in evidenza le posizioni di colore giallo. </li><li> Quindi ripetere la stessa operazione per le posizioni A2 a E3, per selezionare quelle posizioni per le misure del campione. </li><li> Ora, caricare il file di Comma Separated variabile (csv) preparati in anticipo, le prescrizioni e le informazioni del campione. </li></ul> Clicca sul &quot;Bene informazioni&quot; scheda della &quot;Finestra Setup MPR&quot;, come mostrato nella Figura 8. <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="Figura 8" /> Figura 8. MPR finestra di installazione, scheda Informazioni Bene. <ul><li> Quindi fare clic sul <img src="/files/ftp_upload/2465/2465icon5.jpg" alt="Icona 5" /> pulsante. Una finestra di Windows Explorer che si aprirà come mostrato nella Figura 9, che permette la localizzazione del &quot;Bene information.csv&quot; file e il caricamento. <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="Figura 9" /> Figura 9. Caricamento ben campione. Dopo aver caricato il &quot;Bene informazione&quot; del file, le &quot;informazioni Well&quot; sarà come mostrato nella Figura 10 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="Figura 10" /> Figura 10. Loaded informazioni ben campione. </li></ul></li></ol></li></ol> 2. PicoGreen saggio Preparazione del campione <ol><li> Preparare 1X tampone TE (10 mM Tris-HCl, 1 mM EDTA) con l&#39;aggiunta di 1 mL di 20 X TE Buffera 19 ml di dI H 2 O. </li><li> Preparare la soluzione reagente PicoGreen aggiungendo 50 microlitri 200 X reagente PicoGreen a 9,95 ml di buffer di 1 X TE. </li><li> Preparare gli standard (Lambda dsDNA) facendo diluizioni seriali di magazzino dsDNA (100 mg / mL). In primo luogo preparare una soluzione di 50 mg / mL dsDNA aggiungendo 25 l di magazzino dsDNA (100 mg / ml) e 25 microlitri 1 x tampone TE. Preparare quindi le soluzioni standard con l&#39;aggiunta di volumi indicati di dsDNA e buffer come elencati nella tabella seguente: <table border="1"><tbody><tr><td> Concentrazione di dsDNA </td><td> Volume di dsDNA </td><td> Volume di tampone </td></tr><tr><td> 1 mg / mL </td><td> 20 l di 50 mg / mL dsDNA </td><td> 980 microlitri 1 X TE Buffer </td></tr><tr><td> 0,1 mg / ml </td><td> 100 l di 1 mg / mL dsDNA </td><td> 900 microlitri 1 X TE Buffer </td></tr><tr><td> 0,01 mg / ml </td><td> 100 l di 0,1 mcg / mL dsDNA </td><td> 900 microlitri 1 X TE Buffer </td></tr><tr><td> 0,001 mg / ml </td><td> 100 l di 0,01 mg / mL dsDNA </td><td> 900 microlitri 1 X TE Buffer </td></tr></tbody></table></li></ol> 3. Misura degli standard e fluorescenza del campione <ol><li> Pipettare 150 ml di ogni standard nei pozzetti A1-E1 di una piastra 96 ​​pozzetti, per Figura 7, e secondo la seguente tabella: <table border="1"><tbody><tr><td> Bene </td><td> Standard </td></tr><tr><td> A1 </td><td> 1 mg / mL </td></tr><tr><td> B1 </td><td> 0,1 mg / ml </td></tr><tr><td> C1 </td><td> 0,01 mg / ml </td></tr><tr><td> D1 </td><td> 0,001 mg / ml </td></tr><tr><td> E1 </td><td> 1 X TE Buffer </td></tr></tbody></table></li><li> Pipettare 150 campioni microlitri sconosciuto in pozzi F1 per H3, mostrato in figura 7 </li><li> Versare la soluzione PicoGreen preparata al punto 1,2 in un trogolo progettato per multicanale uso pipetta. Utilizzare una pipetta multicanale per fornire 150 ul di soluzione PicoGreen ai pozzetti A1-H3. Mix di soluzioni e standard delicatamente con pipetta. </li><li> Piastra in luogo buio e aspettare 2-5 minuti per la reazione di svilupparsi. </li></ol> 4. Rappresentante Risultati Una buona curva di calibrazione viene valutata utilizzando i diversi parametri forniti dal F-7000 del software su misura degli standard e il calcolo della curva di calibrazione dal software. Il coefficiente di determinazione indica la bontà di adattamento degli standard misurata e la curva di calibrazione calcolati. Quanto più questo valore è a &quot;1&quot;, migliore è la misura del valore misurato e la curva di calibrazione. Se il valore è ben lungi dall&#39;essere &quot;1&quot;, le norme devono essere ri-misurato, preparato ancora una volta, o l&#39;idoneità della curva di calibrazione cambiato. Figura 11 mostra un esempio di una curva di calibrazione con un coefficiente di determinazione calcolato = 0,99993, ottenuto per la quantificazione del DNA a doppia elica con PicoGreen colorante. <img src="/files/ftp_upload/2465/2465fig11.jpg" alt="Figura 11" /> Figura 11. Esempio di curva di calibrazione dsDNA.

<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 e Kendra L. Cox sono dipendenti di Hitachi High Technologies America, che produce lo strumento utilizzato in questo articolo.

Pipettaggio attenzione durante la preparazione dei campioni, così come usando uno spettrofotometro stabile e sensibile fluorescenza è essenziale per ottenere buoni risultati e riproducibili. Nel caso dello spettrofotometro a fluorescenza utilizzata per questo test, si consiglia di utilizzare 300 microlitri volume del campione finale nel lettore per micropiastre. Abbassare il volume diminuisce la sensibilità, e un maggiore volume può causare la contaminazione incrociata tra pozzi.

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

Quantificazione del DNA, specialmente in piccole concentrazioni, è un compito importante, con una vasta gamma di applicazioni biologiche tra cui standard di test di biologia molecolare come la sintesi e la purificazione del DNA, applicazioni diagnostiche come la quantificazione dei prodotti di amplificazione del DNA, e la rilevazione di molecole di DNA in droga preparati. Durante questo video viene mostrata la capacità del spettrofotometro Hitachi F-7000 fluorescenza dotato di un accessorio Micro Reader piastra per eseguire quantificazione dsDNA utilizzando sonde molecolari Quant-it PicoGreen kit di reagenti coloranti. Lo spettrofotometro a fluorescenza F-7000 offre alta sensibilità e le misure ad alta velocità. Si tratta di un sistema altamente flessibile in grado di misurare la fluorescenza, luminescenza e fosforescenza. Modalità di misurazione sono disponibili diversi, tra cui lunghezza d&#39;onda di scansione, scansione del tempo, fotometria e 3-D di misura scansione. Lo spettrofotometro ha una sensibilità nel range di 50 picomoles di fluorescina quando si utilizza un volume di 300 microlitri del campione in micropiastra, ed è in grado di misurare la velocità di scansione di 60.000 nm / minuto. Dispone inoltre di un ampio range dinamico fino a 5 ordini di grandezza, che consente l&#39;utilizzo di curve di calibrazione in un ampio range di concentrazioni. Il sistema ottico utilizza tutte le ottiche riflettenti per la massima energia e sensibilità. La gamma di lunghezze d&#39;onda di serie è da 200 a 750 nm, e può essere estesa a 900 nm quando si usa uno dei fotomoltiplicatori opzionale vicino infrarosso. Il sistema permette il controllo della temperatura opzionale per il lettore di piastre 5-60 gradi Celsius usando un esterno opzionale circolatore controllato la temperatura del liquido. Il lettore di micropiastre consente l&#39;utilizzo di 96 micropiastre bene, e la velocità di misura per 96 pozzetti è meno di 60 secondi quando si utilizza la modalità cinetica. Controlli software per l&#39;F-7000 e micropiastre Reader sono anche molto flessibili. I campioni possono essere impostati sia in formato colonna o riga, e qualsiasi combinazione di pozzi possono essere scelti per la misura del campione. Questo consente un utilizzo ottimale della micropiastra. Inoltre, il software consente l&#39;importazione micro configurazioni di esempio piatto creato in Excel e salvato in valori separati da virgola, o in formato &quot;csv&quot;. Micropiastre configurazioni di misura possono essere salvate e richiamate dal software per comodità e maggiore produttività. Risultati dei dati può essere emesso un rapporto standard, in Excel, o di un programma facoltativo Report Generator.

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

Dimostrazione di quantificazione del dsDNA utilizzando sonde molecolari PicoGreen colorante e Hitachi F-7000 Spettrofotometro a fluorescenza dotato di un accessorio lettore di micropiastre.

Quantificazione di dsDNA con il F-7000 Hitachi Fluorescenza spettrofotometro e PicoGreen Dye

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

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24.4K Views.  Hitachi High Technologies America. Dimostrazione di quantificazione del dsDNA utilizzando sonde molecolari PicoGreen colorante e Hitachi F-7000 Spettrofotometro a fluorescenza dotato di un accessorio lettore di micropiastre.

Video: 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.

Microvolumi Concentrazione Determinazione delle proteine ​​con il NanoDrop 2000c Spettrofotometro

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

Ricerca

Biologia

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.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

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.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

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.

Determinazione concentrazione di acidi nucleici e proteine ​​Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano

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 Quantum la misurazione del rendimento dei campioni di polvere

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.

Visualizzazione di Caenorhabditis Elegans Strutture cuticola Utilizzando il Dye lipofili Vital, 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.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione 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).

Osservazione e quantificazione dei telomeri e sequenze ripetitive Usando fluorescenza<em&gt; In Situ</em&gt; Ibridazione (FISH) con sonde PNA 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.

Visualizzazione e quantificazione dei tessuti adiposi marroni e beige nei topi utilizzando [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.

Quantificazione della competizione interbatterica utilizzando l'imaging a fluorescenza a singola cellula

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