This work was supported in part by the US National Institutes of Health (R01 HL 111600) and the American Heart Association (12SDG9010015).

Toutes les procédures impliquant des animaux ont été approuvés par le soin et l&#39;utilisation des animaux Comité de l&#39;Université de Californie, Davis, et adhéré à la Guide pour les soins et l&#39;utilisation des animaux de laboratoire publié par les National Institutes of Health. 1. Préparation <ol><li> Préparer deux concentrés (25X) les stocks de solution modifiée de Tyrode à l&#39;avance et conserver à 4 ° C: (1) Stock I (en mM: NaCl 3205, CaCl2 32,5, KCl 117,5, NaH 2 PO 4 29.75, MgCl2 26.25) et (2) Stock II (en mM: NaHCO 3 500). <ol><li> Fraîchement préparer une solution de 2 L Tyrode de la composition suivante (en mM): NaCl 128,2, CaCl 2 1,3, KCl 4,7, MgCl2 1,05, NaH 2 PO 4 1,19, NaHCO 3 20 et glucose 11,1 en combinant 1840 ml d&#39;eau désionisée (DI) l&#39;eau, 80 ml Stock I, 80 ml de stock II, et 4 g de glucose. Ne pas mélanger directement Stock I et II Stock; ajouter lem à 1.840 ml DI pour éviter la précipitation. </li></ol></li><li> Préparer la solution d&#39;achat d&#39;actions de l&#39;excitation-contraction découplant blebbistatin à 10 mg / ml dans anhydre di-méthylsulfoxyde (DMSO) à l&#39;avance et conserver la solution d&#39;achat d&#39;actions à 4 ° C. </li><li> Préparer des solutions mères de colorants fluorescents: (1) RH237 tension sensible colorant (1 mg / ml dans le DMSO) et (2) Indicateur de faible affinité calcium Fluo-5N AM (2 mg / ml dans le DMSO) selon les recommandations du fabricant. Utilisez les préparatifs immédiatement pour éviter la décomposition avec perte subséquente de la capacité de chargement. </li><li> Le premier système de recirculation de la perfusion de Langendorff avec oxygénée (95% de O 2, CO 2 5%) solution de Tyrode. Régler le débit de O 2 / CO 2 pour maintenir le pH de la solution de Tyrode à 7,4 ± 0,05. Utilisez un filtre net en ligne en nylon tissé (taille des pores: 11 pm) pour filtrer en continu la solution de perfusion. <ol><li> Premier système de perfusion à température ambiante, comme loadi ultérieureng de Fluo-5N AM est effectuée à température ambiante. Pour Fluo-5N AM chargement, utiliser un petit volume de recirculation de perfusion (~ 150 ml). Après charge de colorant, utiliser un plus grand volume de liquide de perfusion (1 - 2 L, voir figure 1C). </li></ol></li><li> Tournez sur le système d&#39;acquisition de données et de préparer pour la surveillance en continu de l&#39;ECG et de la perfusion pression. Préparer le système de surveillance de pression: Brancher un capteur de pression à la ligne de perfusion et de surveiller la pression de perfusion avec un amplificateur de Transbridge. Ajuster la pression de perfusion de base à 0 mm de Hg lorsque le coeur ne soit pas connectée au système de perfusion. </li><li> Alignez les caméras de cartographie optique pour assurer un alignement spatiale entre le m V et SR Ca 2+ signaux. <ol><li> Tout d&#39;abord, concentrer les caméras en plaçant une règle ou un objet avec le texte dans le plat de perfusion. Tournez les caméras de cartographie dans un mode de vue en direct et la mise au point jusqu&#39;à ce que le texte est clair et net. Acquérir une image de trame unique de chaque caméra. </li><li> Overlayces images et d&#39;ajuster la transparence de l&#39;image du haut de sorte que les deux images sont visibles. Le texte dans les images doivent se chevaucher exactement. Si pas parfaitement alignés, de régler l&#39;angle du miroir dichroïque ou la position de chaque caméra jusqu&#39;à ce que les deux images sont alignées. </li></ol></li></ol> 2. récolte, de perfusion, et Dye-chargement du Lapin Coeur <ol><li> Fixez le lapin dans un dispositif de retenue de lapin approuvé. Profondément anesthésier via une injection intraveineuse (veine marginale de l&#39;oreille) de pentobarbital sodique (50 mg / kg) et de l&#39;héparine (1000 IU). <ol><li> Lorsque le lapin affiche absence de réflexes nociceptifs, faire une incision cutanée médiane pour exposer le sternum et les côtes. Couper à travers le sternum avec des ciseaux chirurgicaux contondants pointe de la xyphoïde au manubrium. Prenez soin de ne pas endommager le coeur lors de l&#39;ouverture du sternum. Étaler les côtes pour exposer le coeur. </li><li> Exciser rapidement l&#39;ensemble du bloc cœur-poumon en coupant rapidement tous les navires et les tissus conjonctifs. Immediadiatement placer le bloc cœur-poumon dans la solution glacée Tyrode. </li></ol></li><li> Situer l&#39;aorte pour perfusion rétrograde. Couper l&#39;aorte ascendante juste en amont de trois branches de la crosse aortique. Cathétériser l&#39;aorte à une canule de 8 G connecté au système de perfusion. Utilisez un morceau de suture USP 0 de soie pour sécuriser l&#39;aorte sur la canule. </li><li> Disséquer soigneusement la trachée, poumons, et la graisse épicardique du coeur. Avec une pince et des ciseaux pointus de dissection, localisez la valve mitrale et soigneusement d&#39;endommager ou de retirer un dépliant pour prévenir la congestion de la solution dans le ventricule gauche. </li><li> Immerger le coeur dans la chambre de perfusion verre chemisé horizontalement avec la surface antérieure du cœur vers le haut pour l&#39;imagerie. Fixer la canule à un morceau de Sylgard avec les repères en forme de U sur le fond de la coupelle de silicium pour empêcher le mouvement du cœur au cours de la cartographie (figure 1D). Si désiré, ajouter une petite épingle d&#39;insectes à la pointe du cœur pour stabilisation. </li><li> Position électrocardiogramme (ECG) électrodes dans le bain sur le côté droit et gauche du cœur. Immerger entièrement les électrodes dans le bain et veiller à ce qu&#39;ils ne sont pas en contact avec la surface du cœur de fournir un ECG de volume menée analogue à une configuration plomb I. Vérifier un plomb je ECG morphologie normale. Ajuster le flux (25 - 35 ml / min) pour s&#39;assurer que la pression aortique est maintenue à 60 - 70 mm de Hg. </li><li> Éteignez les lumières de la pièce et d&#39;ajouter de 0,3 à 0,6 ml de solution stock blebbistatin (étape 1.2) au perfusat (concentration finale 10 - 20 uM). <ol><li> Éteignez les lumières des chambres pour éviter photoinactivation de blebbistatin. Si nécessaire, un petit coup de projecteur ou projecteur peuvent être utilisés pour fournir un éclairage de la tâche. Remarque: blebbistatin est un inhibiteur d&#39;ATPase de la myosine et un découpleur excitation-contraction. Parce Fluo-5N chargement nécessite des conditions prolongées (60 min) RT (hypothermie) (voir les étapes 2.8 - 2.10), la production d&#39;énergie cardiaque pourrait être compromise. Therefore, plus de blebbistatin dans le perfusat avant charge de colorant peut réduire la demande d&#39;énergie 16 et d&#39;éliminer les artefacts de mouvement pendant les enregistrements optiques ultérieurs 17. </li></ol></li><li> Lorsque la contraction du cœur a cessé (10 - 15 min, vérifié sur l&#39;enregistrement de la pression aortique), basculer vers le système de recirculation perfusion de petit volume (voir l&#39;étape 1.4.1). </li><li> Préparer et charger solution de chargement Fluo-5N AM: Ajouter 0,25 ml de Fluo-5N AM solution stock (étape 1.3) à 0,25 ml chaude 20% de Pluronic F127. Ajouter 0,5 ml de solution de Tyrode chaud au mélange, bien mélanger et ajouter au système de recirculation perfusion de petit volume. </li><li> Surveiller en permanence la pression de perfusion et de l&#39;ECG et de régler le débit si nécessaire. Dye chargement dure environ 1 heure à température ambiante. 45 min après le chargement a commencé, tourner sur le bain d&#39;eau circulant pour commencer le réchauffement du système de perfusion et de perfusion à 37 ° C. </li><li> Après 60 min de Fluo-5N AM chargement, switch la perfusion à l&#39;ensemble du système de recirculation de perfusion de volume (voir l&#39;étape 1.4). </li><li> Diluer 50 pi de tension sensible solution colorant RH237 de stock (étape 1.3) dans 1 ml de la solution de Tyrode tiède et ajoutez lentement (~ 5 minutes) dans une proximale de l&#39;orifice d&#39;injection de la canule. </li></ol> 3. cartographie optique <ol><li> Pendant les derniers moments de charge de colorant, positionner le coeur et concentrer les caméras de cartographie optique pour assurer le champ approprié de vue de l&#39;expérience. </li><li> Si désiré, placer une électrode de stimulation bipolaire sur la surface épicardique du cœur pour la stimulation. </li><li> Placez un plastique ou en verre lamelle sur la surface de la chambre de perfusion pour réduire l&#39;artefact de mouvement sur la surface du liquide qui peut être présent en raison de la recirculation du liquide de perfusion. Remarque: Comme alternative à une lamelle, utilisez un couvercle de Petri plat propre plastique de 50 mm. </li><li> Concentrer les guides de lumière pour éclairer uniformément la surface du cœur avec eXCitation lumière. Utilisez diode électroluminescente bleue (LED) sources de lumière et de filtrer davantage la lumière avec un 475-495 nm filtre passe-bande (figure 1A). </li><li> Recueillir fluorescence émise avec un macroscope et deux complémentaires métal-oxyde-semiconducteur (CMOS) caméras de cartographie optique. Diviser la lumière émise avec un miroir dichroïque à 545 nm. <ol><li> Filtre passe-long de la longueur d&#39;onde fragment, contenant le signal de V m, à 700 nm. -Filtre passe-bande de longueur d&#39;onde la plus courte fragment, contenant le SR signal de Ca 2+, 502 à 534 nm (figure 1A). Régler la fréquence d&#39;acquisition de données optiques de 0,5 à 1 kHz. </li></ol></li><li> Pour chaque enregistrement optique, d&#39;abord lancer le protocole de stimulation souhaité, allumez la lumière d&#39;excitation, et de recueillir: 1 - 4 sec des données. Si désiré, synchroniser l&#39;acquisition des protocoles et des données stimulation. Remarque: Il est impossible de re-charger le Fluo-5N AM, donc le rapport signal sur bruit (SNR) sera decreasoi tout au long de l&#39;expérience en raison de teindre les fuites de la SR. Limitez protocole expérimental à 1 - 2 h pour assurer des valeurs élevées de SNR. </li><li> À la suite de l&#39;expérience, laver le système de perfusion dans la séquence de l&#39;eau DI, 70% d&#39;alcool réactif, et de nouveau avec de l&#39;eau DI. </li><li> Filtre à chaque jeu de données avec un filtre gaussien spatial (rayon de 3 pixels) en utilisant des progiciels disponibles dans le commerce selon le protocole du fabricant. </li><li> Si nécessaire, aligner spatialement la m V et SR Ca 2+ ensembles de données. Comme à l&#39;étape 1.6, superposer des images de chaque caméra et vérifier que les structures anatomiques du coeur (ie., Vasculaire coronaire, les bords des oreillettes ou les ventricules) ou d&#39;autres objets dans le champ de vision (par exemple, la canule, l&#39;électrode de stimulation) exactement chevauchement. Si non, alignement spatial des ensembles de données est nécessaire. </li><li> Décaler l&#39;image supérieure transparente dans le x et y direction jusqu&#39;à ce que le chevauchement exacte est atteinte, en prenant note du nombre de pixelsl&#39;image doit être décalée dans chaque direction. L&#39;ensemble des données correspondant à l&#39;image du haut (soit V m ou SR Ca 2+) doit alors être déplacé par le nombre déterminé de pixels dans chaque direction pour assurer un alignement précis entre le m V et SR Ca 2+ ensembles de données. </li></ol>

<ol>
	<li>Herron, T. J., Lee, P., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+voltage+and+calcium+in+cardiac+cells.">Optical imaging of voltage and calcium in cardiac cells.</a> tissues. Circulation research. 110, 609-623 (2012).</li><li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circulation research. 95, 21-33 (2004).</li><li>Lou, Q., Li, W., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+optical+mapping+of+the+Langendorff-perfused+rabbit+heart.">Multiparametric optical mapping of the Langendorff-perfused rabbit heart.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Choi, B. R., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+maps+of+optical+action+potentials+and+calcium+transients+in+guinea-pig+hearts:+mechanisms+underlying+concordant+alternans.">Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.</a> The Journal of physiology. 529 (Pt 1), 171-188 (2000).</li><li>Kong, W., Fast, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dye+affinity+in+optical+measurements+of+Cai(2+)+transients+in+cardiac+muscle.+American+journal+of+physiology.">The role of dye affinity in optical measurements of Cai(2+) transients in cardiac muscle. American journal of physiology.</a> Heart and circulatory physiology. 307, H73-H79 (2014).</li><li>Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Excitation-Contraction Coupling and Cardiac Contractile Force. , (2001).</li><li>Bers, D. M., Shannon, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+movements+inside+the+sarcoplasmic+reticulum+of+cardiac+myocytes.">Calcium movements inside the sarcoplasmic reticulum of cardiac myocytes.</a> Journal of molecular and cellular cardiology. , (2013).</li><li>Knollmann, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+roles+of+calsequestrin+and+triadin+in+cardiac+muscle.">New roles of calsequestrin and triadin in cardiac muscle.</a> The Journal of physiology. 587, 3081-3087 (2009).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+calsequestrin+regulation+of+single+cardiac+ryanodine+receptor+in+normal+and+pathological+conditions.">Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions.</a> The Journal of general physiology. 142, 127-136 (2013).</li><li>Diaz, M. E., O'Neill, S. C., Eisner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcoplasmic+reticulum+calcium+content+fluctuation+is+the+key+to+cardiac+alternans.">Sarcoplasmic reticulum calcium content fluctuation is the key to cardiac alternans.</a> Circulation research. 94, 650-656 (2004).</li><li>Shannon, T. R., Guo, T., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++scraps:+local+depletions+of+free+[Ca2+]+in+cardiac+sarcoplasmic+reticulum+during+contractions+leave+substantial+Ca2++reserve.">Ca2+ scraps: local depletions of free [Ca2+] in cardiac sarcoplasmic reticulum during contractions leave substantial Ca2+ reserve.</a> Circulation research. 93, 40-45 (2003).</li><li>Kornyeyev, D., Reyes, M., Escobar, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+Ca(2+)+content+regulates+intracellular+Ca(2+)+release+in+subepicardial+myocytes+of+intact+beating+mouse+hearts:+effect+of+exogenous+buffers.">Luminal Ca(2+) content regulates intracellular Ca(2+) release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers.</a> American journal of physiology. Heart and circulatory physiology. 298, H2138-H2153 (2010).</li><li>Picht, E., DeSantiago, J., Blatter, L. A., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+alternans+do+not+rely+on+diastolic+sarcoplasmic+reticulum+calcium+content+fluctuations.">Cardiac alternans do not rely on diastolic sarcoplasmic reticulum calcium content fluctuations.</a> Circulation research. 99, 740-748 (2006).</li><li>Shkryl, V. M., Maxwell, J. T., Domeier, T. L., Blatter, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractoriness+of+sarcoplasmic+reticulum+Ca2++release+determines+Ca2++alternans+in+atrial+myocytes.+American+journal+of+physiology.">Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes. American journal of physiology.</a> Heart and circulatory physiology. 302, H2310-H2320 (2012).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+sarcoplasmic+reticulum+Ca2++in+the+intact+heart:+ryanodine+receptor+refractoriness+during+alternans+and+fibrillation.">Optical mapping of sarcoplasmic reticulum Ca2+ in the intact heart: ryanodine receptor refractoriness during alternans and fibrillation.</a> Circulation research. 114, 1410-1421 (2014).</li><li>Wengrowski, A. M., Kuzmiak-Glancy, S., Jaimes, R., Kay, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADH+Changes+During+Hypoxia,+Ischemia,+and+Increased+Work+Differ+Between+Isolated+Heart+Preparations.">NADH Changes During Hypoxia, Ischemia, and Increased Work Differ Between Isolated Heart Preparations.</a> American journal of physiology. Heart and circulatory physiology. , (2013).</li><li>Fedorov, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+blebbistatin+as+an+excitation-contraction+uncoupler+for+electrophysiologic+study+of+rat+and+rabbit+hearts.">Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts.</a> Heart rhythm : the official journal of the Heart Rhythm Society. 4, 619-626 (2007).</li><li>Myles, R. C., Wang, L., Kang, C., Bers, D. M., Ripplinger, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+beta-adrenergic+stimulation+overcomes+source-sink+mismatch+to+generate+focal+arrhythmia.">Local beta-adrenergic stimulation overcomes source-sink mismatch to generate focal arrhythmia.</a> Circulation research. 110, 1454-1464 (2012).</li><li>Marks, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+cycling+proteins+and+heart+failure:+mechanisms+and+therapeutics.">Calcium cycling proteins and heart failure: mechanisms and therapeutics.</a> The Journal of clinical investigation. 123, 46-52 (2013).</li><li>Cutler, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+sarcoplasmic+reticulum+Ca2++ATPase+2a+gene+delivery+to+restore+electrical+stability+in+the+failing+heart.">Targeted sarcoplasmic reticulum Ca2+ ATPase 2a gene delivery to restore electrical stability in the failing heart.</a> Circulation. 126, 2095-2104 (2012).</li></ol>

The authors declare that they have no competing financial interests.

Les clés du succès Fluo-5N charge de colorant sont la configuration recirculation perfusion de petit volume, ce qui permet une concentration élevée Fluo-5N sans avoir besoin de grandes quantités de colorant, la longueur des temps de chargement (1 h), et effectuer le chargement à la température ambiante. Si le chargement est effectué à des températures physiologiques, l&#39;activité enzymatique cellulaire rapidement clive le tag de -AM lorsque le colorant traverse la membrane cellulaire, le piégeage des molé...

Dual optical mapping of intracellular Ca2+ and transmembrane potential (Vm) in the intact Langendorff-perfused heart has become a mainstay of investigations in cardiac electrophysiology, including mechanisms of arrhythmia and excitation-contraction coupling1-4. This approach has provided unprecedented knowledge into normal and abnormal electrophysiology and, importantly, into the bidirectional relationship between Vm and intracellular Ca2+. However, optical mapping of intracellular Ca2+ with high-affinity fluorescent indicators (such as Rhod-2 and Fluo-4) only reports on bulk changes in intracellular Ca2+ and is unable to distinguish whether these changes are due to transmembrane Ca2+ flux, release and reuptake into intracellular stores, or in most instances, some combination of both. Furthermore, high-affinity Ca2+ indicators have slow on-off kinetics and may not accurately report rapid changes in Ca2+ concentration5.
Each action potential triggers a rise in intracellular Ca2+, known as the intracellular Ca2+ transient (CaT). In the mammalian heart, approximately 70 &#8211; 90% of the total CaT is due to release of Ca2+ from the sarcoplasmic reticulum (SR) via opening of ryanodine receptors (RyRs)6. Within the SR, approximately half of the total Ca2+ is bound to calsequestrin (CSQ) and other intra-SR buffers7, which play an important role in SR Ca2+ homeostasis8,9. The amount of free SR Ca2+ dictates the driving force for SR Ca2+ release as well as gating of RyR, and therefore has a significant impact on the intracellular CaT. Furthermore, alterations in SR Ca2+ release or reuptake can, in turn, impact Vm via the electrogenic Na+-Ca2+ exchange, which may have arrhythmogenic consequences. Therefore, in addition to the CaT, monitoring of free SR Ca2+ can provide important insights into contractile and electrophysiological dysfunction.
Over the past several years, investigators have made significant advances in the monitoring of SR Ca2+ in isolated cardiac myocytes and from a single location on the intact heart. One such method requires rapid pulses of caffeine to open RyRs and the SR Ca2+ content is then inferred or calculated from the immediate rise in intracellular Ca2+10. Another intriguing approach uses low-affinity Ca2+ indicators, such as Fluo-5N11 or Mag-Fluo412, which bind to free SR Ca2+. These indicators have dissociation constants (Kd) in the range of 10 &#8211; 400 &#956;M and therefore exhibit minimal fluorescence in the cytosol compared to the SR lumen, where the Ca2+ concentration ([Ca2+]SR) is in the millimolar range. Using low-affinity Ca2+ indicators, several aspects of SR Ca2+ cycling have been investigated at the level of the isolated myocyte, including fractional SR Ca2+ release and the mechanisms of Ca2+ alternans13,14. However, in order to fully understand the heterogeneous nature of SR Ca2+ cycling in the intact heart and the role of SR Ca2+ in spatially distinct arrhythmic phenomena, methods for imaging SR Ca2+ across the epicardial surface of the intact heart are required15.
This article describes methodology for dual optical mapping of free SR Ca2+ and Vm in the intact Langendorff-perfused rabbit heart with the low-affinity Ca2+ indicator Fluo-5N. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+.

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S271-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">CaCl2 (2H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">C79-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">KCl</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S217-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MgCl2 (6H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">M33-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaH2PO4 (H2O)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S369-500</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">NaHCO3</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">S233-3</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">D-Glucose</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:151px;">D16-1</td>
			<td style="width:423px;">Component of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">95% O2 5% CO2</td>
			<td style="width:167px;">AirGas</td>
			<td style="width:151px;">carbogen</td>
			<td style="width:423px;">For oxygenation and pH of Tyrode&#39;s solution</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Blebbistatin</td>
			<td style="width:167px;">Tocris Bioscience</td>
			<td style="width:151px;">1760</td>
			<td style="width:423px;">Excitation-contraction uncoupler</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">RH237</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">61018</td>
			<td style="width:423px;">Voltage-sensitive dye</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Fluo-5N AM</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:151px;">F-26915</td>
			<td style="width:423px;">Low-affinity Ca2+ indicator; Alternative: Invitrogen F-14204; Loading must be performed at room temperature</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pluronic F127</td>
			<td style="width:167px;">Biotium</td>
			<td style="width:151px;">59004</td>
			<td style="width:423px;">For Ca2+ indicator loading; Warm until the solutiion is clear before use</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Dimethyl sulphoxide (DMSO)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td style="width:423px;">For dissolving blebbistatin and dyes</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Filter</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:151px;">NY1104700</td>
			<td style="width:423px;">11um in-line filter</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Pressure Transducer</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">BLPR2</td>
			<td style="width:423px;">For measuring perfusion pressure</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Transbridge Transducer Amplifier</td>
			<td style="width:167px;">WPI</td>
			<td style="width:151px;">SYS-TBM4M</td>
			<td style="width:423px;">For transducing/amplifing pressure signal; PowerLab may also be used with appropriate BioAmp</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">PowerLab 26T</td>
			<td style="width:167px;">ADInstruments</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">For continuous recording of pressure and ECG signals</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">THT Macroscope</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Macroscopic optical setup. Details: 0.63x objective (NA=0.31), 2x condensing objective, resultant field of view = 3.1x3.1 cm, depth of focus = ~1.5mm</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">MiCam Ultima-L CMOS</td>
			<td style="width:167px;">SciMedia</td>
			<td style="width:151px;"></td>
			<td style="width:423px;">Optical mapping cameras</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Precision LED Spot Light</td>
			<td style="width:167px;">Mightex</td>
			<td style="width:151px;">PLS-0470-030-15-S</td>
			<td style="width:423px;">LED light source</td>
		</tr>
	</tbody>
</table>

optical mapping of intra-sarcoplasmic reticulum ca2+ and transmembrane potential in the langendorff-perfused rabbit heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

La figure 1A représente un diagramme schématique de la configuration optique pour double m V SR Ca2 + et la cartographie. Avec cette configuration, il ya une séparation spectrale complète de la m V et SR Ca 2+ signaux (figure 1B). Un schéma du système de perfusion à double boucle utilisée pour Fluo-5N colorant chargement est illustrée à la figure 1C. La figure 1D montre l&#39;orientation horizontale ...

Watch this Scientific Journal Video about Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart at JoVE.com

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Cet article décrit le protocole détaillé et l&#39;équipement nécessaire pour une double cartographie optique du potentiel transmembranaire (V m) et d&#39;une connexion intra-réticulum sarcoplasmique (SR) Ca 2+ dans le coeur de lapin Langendorff perfusé. Cette méthode permet l&#39;observation directe et la quantification de V m et SR Ca 2+ dynamique dans le coeur intact.

Cartographie optique du commerce intra-réticulum sarcoplasmique Ca<sup&gt; 2+</sup&gt; Et potentiel transmembranaire dans le Lapin Coeur Langendorff perfusé

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

optical-mapping-intra-sarcoplasmic-reticulum-ca2-transmembrane

Research

JoVE Journal

Biology

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

9.1K vues.  University of California Davis.  Cet article décrit le protocole détaillé et l'équipement nécessaire pour une double cartographie optique du potentiel transmembranaire (V m) et d'une connexion intra-réticulum sarcoplasmique (SR) Ca 2+ dans le coeur de lapin Langendorff perfusé. Cette méthode permet l'observation directe et la quantification de V m et SR Ca 2+ dynamique dans le coeur intact. 

Vidéo: Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Entretien et nettoyage du microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Recherche

Biologie

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Cartographie optique des coeurs de rats perfusés de Langendorff

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualiser le cœur battant Drosophile</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Analyse semi-automatisée Heartbeat optique de petits coeurs

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

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

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

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

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

Préparation d&#39;embryons pour la microscopie électronique des Drosophile Tube cardiaque embryonnaire

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

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 ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Évaluation de la distribution spatiale des rayonnements ionisants suivantes γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Test clonogénique: cellules adhérentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Tension et calcium double cartographie de canal optique de Cultured HL-1 auriculaire myocytes monocouche

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.