1. Configuration de l&#39;instrument <ol><li> Allumez le spectrofluorimètre et laisser chauffer pendant 1 heure. </li><li> Utilisation d&#39;Excel créer un fichier avec les normes et les données d&#39;échantillon, comme le montre la figure 1 et l&#39;enregistrer dans Comma Separated Values ​​&quot;CSV&quot;. <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="Figure 1" /> Figure 1. Normes et les données des échantillons. </li><li> Mettre en place fluorimètre comme suit: <ol><li> Cliquez sur le <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="Icône 1" /> bouton, cela ouvrira la fenêtre de méthode d&#39;analyse, montre la figure 2. <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="Figure 2" /> Figure 2. Fenêtre méthode d&#39;analyse, onglet Général. Effectuez les sélections suivantes: <ul><li> Dans la &quot;mesure&quot;, sélectionnez Photométrie du menu déroulant. </li><li> En champ &quot;Opérateur&quot;, entrez le nom de l&#39;opérateur approprié. </li><li> Il n&#39;est pas nécessaire d&#39;entrer des informations dans le «Règlement» le terrain. Cette option est sélectionnée automatiquement par le logiciel. </li><li> Le &quot;prélèvement&quot; champ est inactif lorsque vous utilisez le lecteur de microplaques. </li><li> Saisissez tout commentaire que vous pourriez inclure dans le rapport de la champ &quot;Commentaires&quot;. </li><li> Dans le &quot;Accessoire&quot;, entrez les informations relatives aux accessoires utilisés pour ce test. </li><li> Notez les boutons sur le côté droit de la fenêtre. </li><li> Le bouton &quot;Charger&quot; permet de charger les méthodes d&#39;analyse précédemment enregistré. </li><li> Le bouton &quot;Sauvegarder&quot; permet la mise à jour d&#39;un ensemble de paramètres de test ou d&#39;une méthode d&#39;analyse préalablement enregistrés et chargés, en utilisant le même nom. </li><li> Le &quot;Enregistrer sous&quot; bouton permet d&#39;enregistrer un nouvel ensemble de paramètres de test ou d&#39;une méthode d&#39;analyse sous un autre nom désiré. </li></ul> Cliquez maintenant sur ​​le &quot;quantification&quot; onglet, voir la figure 3 et effectuez les sélections suivantes. <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="Figure 3" /> Figure 3. Fenêtre méthode d&#39;analyse, onglet quantification. <ul><li> Dans le &quot;type de quantification&quot;, sélectionnez onde. Ceci indique la lecture de fluorescence seront effectués en utilisant la longueur d&#39;onde sélectionnée. </li><li> Dans le «type d&#39;étalonnage&quot;, sélectionnez une vue st. </li><li> Dans le champ &quot;Nombre de longueurs d&#39;onde&quot;, sélectionnez 1. </li><li> Dans l &#39;&quot;unité de concentration&quot;, entrez ng / mL. </li><li> Laisser les cases non sélectionnées pour &quot;Calibrage manuel» et «courbe de force grâce à zéro&quot;. </li><li> Dans le &quot;chiffre après la virgule&quot;, sélectionnez 2. </li><li> Dans la &quot;limite inférieure de concentration&quot;, entrez 0. </li><li> Dans le &quot;champ de concentration limite supérieure, entrez 10000. </li></ul> Cliquez maintenant sur ​​«l&#39;Instrument» onglet et effectuez les sélections suivantes. <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="Figure 4" /> Figure 4. Fenêtre de méthode d&#39;analyse, onglet Instrument <ul><li> Dans le mode &quot;données&quot; de terrain, sélectionnez fluorescence. </li><li> La «vitesse à découper« champ est inactif en ce moment, il n&#39;est utilisé que pour des mesures de phosphorescence. </li><li> Dans le mode &quot;Wavelength&quot;, sélectionnez la fois fixe WL. </li><li> Dans le &quot;EX / WL1&quot; entrez 480nm. </li><li> Dans le «EM / WL1&quot; entrez 520nm. </li><li> Tous les autres champs sous EX et EM sont inactifs en ce moment. </li><li> Dans la &quot;fente EX&quot; champ de sélectionner 5.0nm du menu déroulant. </li><li> Dans le «EM fente&quot; sur le terrain de sélectionner 5.0nm du menu déroulant. </li><li> Laisser désélectionné la case en face de la &quot;tension PMT 1-1000V&quot; sur le terrain. </li><li> Dans la &quot;tension PMT&quot;, sélectionnez 700V à partir du menu déroulant. </li><li> Dans le «calc Auto statistique. Numéro de&quot; champ de sélectionner 2. </li><li> Dans la «Réplique» champ de sélectionner 1. </li><li> Dans le «Temps d&#39;intégration&quot; champ de sélectionner 0.1s. &lt;/ Li&gt; <li> Dans le &quot;retard&quot; entrez ou sélectionnez 0. </li></ul> Cliquez sur l&#39;onglet &quot;Moniteur&quot;. <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="Figure 5" /> Figure 5. Fenêtre de méthode d&#39;analyse, onglet Moniteur. <ul><li> Dans «l&#39;axe Y&quot;, &quot;Max:&quot;, sélectionnez 10000 </li><li> Dans «l&#39;axe Y&quot;, &quot;Min:« champ, sélectionnez 0. </li><li> Sélectionnez la case à gauche du &quot;traitement de données ouvertes après l&#39;acquisition des données&quot;. </li><li> Laisser désélectionné la case à la gauche du &quot;rapport d&#39;impression après l&#39;acquisition des données&quot;, sauf si vous voulez un rapport qui sera imprimé automatiquement après les lectures sont terminées. </li></ul> Cliquez sur l&#39;onglet &quot;Report&quot;, voir la figure 6. <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="Figure 6" /> Figure 6. Fenêtre de méthode d&#39;analyse, onglet Rapport. <ul><li> Dans la &quot;sortie&quot;, sélectionnez Imprimer rapport du menu déroulant. Vous pouvez également sélectionner &quot;Utiliser Microsoft Excel» si vous voulez que les données exportées vers Excel, ou la «feuille Générateur Utilisez Imprimer» si vous utilisez le complément optionnel sur Report Generator programme qui permet de générer des rapports personnalisés de fait. </li><li> Cliquez sur les cases des éléments que vous voulez être inclus dans le rapport. </li><li> Pour enregistrer tous les paramètres sélectionnés dans les différents onglets de la «Méthode d&#39;analyse&quot; fenêtre, cliquez à nouveau sur l&#39;onglet &quot;Général&quot; et de les enregistrer sous un nom de fichier et dans un endroit où vous pourrez les trouver pour une utilisation future, et cliquez sur le &quot; OK &quot;pour fermer cette fenêtre. </li><li> Ceci achève la mise en place des paramètres de test instrument. </li><li> Maintenant, nous allons mettre en place le lecteur de microplaques. </li><li> Cliquez sur le <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="Icône 2" /> bouton, ce qui portera la fenêtre de configuration du MPR sur le dessus. </li></ul> Sélectionner des puits pour les normes et les échantillons dans la configuration de MPR fenêtre / onglet Plate, comme le montre la Figure 7. <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="Figure 7" /> Figure 7. Fenêtre d&#39;installation MPR, onglet Plate. <ul><li> Cliquez sur la position A1 et faites glisser la souris pour E1, puis cliquez sur le <img src="/files/ftp_upload/2465/2465icon3.jpg" alt="Icône 3" /> bouton. Cela permet de sélectionner les postes à être utilisée pour les normes. Ces positions seront surlignés en couleur verte. </li><li> Maintenant nous avons besoin pour sélectionner F1 postes à H3 pour les échantillons. Cliquez et faites glisser la souris à partir de F1 position, à la position H3 et puis cliquez sur le <img src="/files/ftp_upload/2465/2465icon4.jpg" alt="Icône 4" /> bouton, cela va mettre en évidence les positions de couleur jaune. </li><li> Ensuite, répétez la même opération pour des postes A2 à l&#39;E3, pour sélectionner les postes pour les mesures d&#39;échantillons. </li><li> Maintenant, nous allons charger le fichier Comma Separated Variable (CSV) préparé à l&#39;avance avec les normes et les informations de l&#39;échantillon. </li></ul> Cliquez sur &quot;information bien&quot; de l&#39;onglet &quot;Fenêtre de configuration du MPR», comme le montre la Figure 8. <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="Figure 8" /> Figure 8. Fenêtre d&#39;installation MPR, onglet Informations bien. <ul><li> Puis cliquez sur le <img src="/files/ftp_upload/2465/2465icon5.jpg" alt="Icône 5" /> bouton. Une fenêtre de l&#39;explorateur Windows s&#39;ouvre qui, comme le montre la figure 9, ce qui permettra à localiser la «Eh bien information.csv&quot; fichier et de le charger. <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="Figure 9" /> Figure 9. Chargement des informations puits d&#39;échantillon. Après le chargement du &quot;information bien&quot; fichier, &quot;l&#39;information Well&quot; sera comme le montre la figure 10 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="Figure 10" /> Figure 10. Chargé d&#39;information de l&#39;échantillon ainsi. </li></ul></li></ol></li></ol> 2. Préparation Picogreen échantillon d&#39;essai <ol><li> Préparer 1X tampon TE (10 mM Tris-HCl, 1 mM EDTA) en ajoutant 1 ml de tampon 20 X TEà 19 ml dI H 2 O. </li><li> Préparer la solution de réactif PicoGreen en ajoutant 50 pl 200 X PicoGreen réactif à 9,95 ml de tampon 1 X TE. </li><li> Préparer des normes (Lambda ADNdb) en faisant des dilutions successives du stock ADNdb (100 pg / mL). D&#39;abord préparer une solution de 50 ug / ml ADNdb en ajoutant 25 uL de stock ADNdb (100 pg / ml) et 25 uL 1 x tampon TE. Puis la préparation des solutions standard en ajoutant les volumes indiqués d&#39;ADN double brin et un tampon comme indiqué dans le tableau ci-dessous: <table border="1"><tbody><tr><td> Concentration des ADNdb </td><td> Volume d&#39;ADN double brin </td><td> Volume de tampon </td></tr><tr><td> 1 ug / ml </td><td> 20 ul de 50 ug / ml ADNdb </td><td> 980 uL 1 X tampon TE </td></tr><tr><td> 0,1 pg / ml </td><td> 100 uL de 1 pg / mL ADNdb </td><td> 900 pi X 1 tampon TE </td></tr><tr><td> 0,01 pg / ml </td><td> 100 uL de 0,1 pg / mL ADNdb </td><td> 900 pi X 1 tampon TE </td></tr><tr><td> 0,001 g / mL </td><td> 100 ul de 0,01 pg / mL ADNdb </td><td> 900 pi X 1 tampon TE </td></tr></tbody></table></li></ol> 3. Mesure des normes et de fluorescence de l&#39;échantillon <ol><li> Pipeter 150 uL de chaque norme dans les puits A1-E1 d&#39;une plaque de 96 puits, par la figure 7, et selon le tableau suivant: <table border="1"><tbody><tr><td> Eh bien </td><td> Standard </td></tr><tr><td> A1 </td><td> 1 ug / ml </td></tr><tr><td> B1 </td><td> 0,1 pg / ml </td></tr><tr><td> C1 </td><td> 0,01 pg / ml </td></tr><tr><td> D1 </td><td> 0,001 g / mL </td></tr><tr><td> E1 </td><td> 1 X tampon TE </td></tr></tbody></table></li><li> Pipeter 150 uL échantillons inconnus dans des puits F1 à H3, illustré à la figure 7 </li><li> Verser la solution PicoGreen préparés à l&#39;étape 1.2 dans une auge conçu pour une utilisation pipette multi-canaux. Utiliser une pipette multi-canaux pour livrer 150 uL solution PicoGreen aux puits A1-H3. Mélanger la solution et les normes doucement avec la pipette. </li><li> Placez la plaque dans la zone sombre et attendre 2-5 minutes pour une réaction à se développer. </li></ol> 4. Les résultats représentatifs Une courbe de calibration de bons est évaluée en utilisant les différents paramètres fournis par le logiciel F-7000 sur la mesure des normes et le calcul de la courbe d&#39;étalonnage par le logiciel. Le coefficient de détermination indique la qualité de l&#39;ajustement des normes mesurées et la courbe de calibration calculé. Le plus proche de cette valeur est à &quot;1&quot;, le mieux l&#39;ajustement de la valeur mesurée et la courbe de calibration. Si la valeur est loin d&#39;être «1», les normes doivent être ré-évalués, préparés à nouveau, ou l&#39;aptitude de la courbe de calibration changé. La figure 11 montre un exemple d&#39;une courbe d&#39;étalonnage avec un coefficient de détermination calculée = 0,99993, obtenus pour la quantification des ADN double brin à l&#39;aide PicoGreen colorant. <img src="/files/ftp_upload/2465/2465fig11.jpg" alt="Figure 11" /> Figure 11. Exemple de courbe de calibration ADNdb.

<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 et Kendra L. Cox sont employés par Hitachi America Technologies Haute qui produit l&#39;instrument utilisé dans cet article.

Pipetage prudent lors de la préparation des échantillons, ainsi que l&#39;aide d&#39;un spectrophotomètre à fluorescence stable et sensible est essentielle pour obtenir de bons résultats et reproductibles. Dans le cas du spectrophotomètre de fluorescence utilisé pour ce test, il est suggéré d&#39;utiliser le volume 300 uL de l&#39;échantillon final dans le lecteur de microplaques. Baisser le volume diminue la sensibilité, et un plus grand volume peut provoquer une contamination croisée entre les puits.

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		<th>Catalogue Number</th>
		<th>Comment</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>
<tr>
<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>
<tr>
<td>Microplate Reader Accessory for F-7000</td>
<td>Equipment</td>
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<td>5J0-0139</td>
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quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

La quantification de l&#39;ADN, en particulier dans les petites concentrations, est une tâche importante avec un large éventail d&#39;applications biologiques, y compris la norme tests de biologie moléculaire telles que la synthèse et la purification de l&#39;ADN, les applications diagnostiques telles que la quantification des produits d&#39;amplification d&#39;ADN, et la détection de molécules d&#39;ADN à la drogue préparations. Durant cette vidéo, nous allons démontrer la capacité du spectrophotomètre Hitachi F-7000 fluorescence équipé d&#39;un accessoire lecteur de plaque d&#39;effectuer une quantification Micro ADNdb utilisant Molecular Probes Quant-on kit PicoGreen réactif colorant. Le spectrophotomètre F-7000 offre une haute sensibilité de fluorescence et les mesures à haute vitesse. C&#39;est un système très flexible capable de fluorescence de mesure, la luminescence, et la phosphorescence. Plusieurs modes de mesure sont disponibles, y compris les longueurs d&#39;onde, balayage temps, la photométrie et en 3-D de balayage de mesure. Le spectrophotomètre a une sensibilité de l&#39;ordre de 50 picomoles de fluorescéine en utilisant un volume d&#39;échantillon 300 ul de la microplaque, et est capable de mesurer des vitesses de numérisation de 60.000 nm / minute. Il a également une large gamme dynamique allant jusqu&#39;à 5 ordres de grandeur qui permet l&#39;utilisation des courbes de calibrage sur une large gamme de concentrations. Le système optique utilise toutes les optiques réfléchissants pour un maximum d&#39;énergie et de sensibilité. La gamme de longueur d&#39;onde est de 200 à 750 nm, et peut être étendue à 900 nm en utilisant l&#39;un des proches option photomultiplicateurs infrarouge. Le système permet un contrôle de température en option pour le lecteur plaque de 5 à 60 degrés Celsius en utilisant une option de la température extérieure circulateur liquide. Le lecteur de microplaques permet l&#39;utilisation de microplaques 96 puits, et la mesure de la vitesse de 96 puits est inférieur à 60 secondes lorsque vous utilisez le mode cinétique. Logiciel de contrôle pour le F-7000 et lecteur de microplaques sont également très flexible. Les échantillons peuvent être mis dans des formats colonne ou une ligne non plus, et toute combinaison de puits peuvent être choisis pour les mesures de l&#39;échantillon. Cela permet une utilisation optimale de la microplaque. De plus, le logiciel permet d&#39;importer des exemples de configurations de micro plaque créés dans Excel et sauvegardées dans des valeurs séparées par des virgules, ou &quot;CSV&quot;. Configurations microplaques de mesure peuvent être sauvegardés et rappelés par le logiciel pour plus de commodité et une productivité accrue. Des résultats Les données peuvent être sorties d&#39;un rapport standard, au format Excel, ou à un programme générateur de rapport facultatives.

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

Démonstration de la quantification des ADN double brin à l&#39;aide Molecular Probes PicoGreen colorant et spectrophotomètre Hitachi fluorescence F-7000 équipé d&#39;un accessoire lecteur de microplaques.

Quantification des ADNdb utilisant le Hitachi F-7000 Spectrophotomètre de fluorescence et de 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

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Biology

24.4K vues.  Hitachi High Technologies America. Démonstration de la quantification des ADN double brin à l'aide Molecular Probes PicoGreen colorant et spectrophotomètre Hitachi fluorescence F-7000 équipé d'un accessoire lecteur de microplaques.

Vidéo: 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.

Détermination microvolume concentration de protéines en utilisant le spectrophotomètre NanoDrop 2000c

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

Recherche

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.

Imagerie time-lapse de la mitose après transfection de 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.

Quantification des γH2AX Foci en réponse aux radiations ionisantes

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.

Détermination de la concentration des acides nucléiques et des protéines en utilisant le micro-volume Bio-spec Spectrophotomètre 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 mesure du rendement quantique des échantillons de poudre

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.

Visualisation des Caenorhabditis elegans</emOuvrages cuticules> Utilisation du colorant lipophile 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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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).

Observation et la quantification des télomères et des séquences répétitives utilisant Fluorescence<em&gt; In Situ</em&gt; Hybridation (FISH) avec PNA Sondes en<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.

Visualisation et quantification des tissus adipeux bruns et beiges chez la souris à l’aide de l’imagerie micro-PET/MR [18F]FDG

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.

Quantification de la concurrence interbactérienne à l’aide de l’imagerie par fluorescence unicellulaire

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