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

Tutte le procedure che coinvolgono gli animali sono stati approvati dalla cura e l&#39;uso Comitato Animale dell&#39;Università di California, Davis, e aderito alla Guida per la cura e l&#39;uso di animali da laboratorio pubblicato dal National Institutes of Health. 1. Preparazione <ol><li> Preparare due (25X) le scorte concentrati di soluzione Tyrode modificato in anticipo e conservare a 4 ° C: (1) Stock I (in mm: NaCl 3205, CaCl 2 32.5, KCl 117,5, NaH 2 PO 4 29.75, MgCl 2 26.25) e (2) Stock II (in mM: NaHCO3 500). <ol><li> Appena preparare la soluzione di 2 L Tyrode della seguente composizione (in mM): NaCl 128,2, CaCl 2 1,3, KCl 4,7, MgCl 2 1.05, NaH 2 PO 4 1.19, NaHCO3 20 e glucosio 11,1 combinando 1840 ml di acqua deionizzata (DI) acqua, 80 ml Stock I, 80 ml di Fotografico II, e 4 g di glucosio. Non miscelare direttamente I Stock e della II; aggiungere ilm 1.840 ml DI per evitare la precipitazione. </li></ol></li><li> Preparare soluzione di riserva della eccitazione-contrazione disaccoppiatore blebbistatin a 10 mg / ml in anidra di-methylsulfoxide (DMSO) in anticipo e conservare la soluzione di riserva a 4 ° C. </li><li> Preparare soluzioni di coloranti fluorescenti: (1) RH237 tensione sensibili dye (1 mg / ml in DMSO) e (2) Indicatore di affinità calcio Fluo-5N AM (2 mg / ml in DMSO) secondo le raccomandazioni del fabbricante. Utilizzare immediatamente i preparativi per evitare la decomposizione, con conseguente perdita di capacità di carico. </li><li> Prime il ricircolo sistema di perfusione Langendorff con ossigenata (95% O 2, 2 5% di CO) soluzione Tyrode. Regolare il flusso di O 2 / CO 2 per mantenere il pH della soluzione di Tyrode a 7,4 ± 0,05. Utilizzare un filtro a rete in filo di nylon tessuto (dimensione dei pori: 11 micron) per filtrare continuamente il perfusato. <ol><li> Prime il sistema di perfusione a temperatura ambiente, come la successiva loading di Fluo-5N AM viene eseguita a temperatura ambiente. Per Fluo-5N AM caricamento, utilizzare un piccolo volume di ricircolo perfusato (~ 150 ml). Dopo colorante carico, utilizzare un volume maggiore di perfusato (1 - 2 L, vedere Figura 1C). </li></ol></li><li> Accendere il sistema di acquisizione dati e di preparare per il monitoraggio continuo di ECG e pressione di perfusione. Preparare il sistema di pressione di monitoraggio: collegare un trasduttore di pressione alla linea di perfusione e monitorare la pressione di perfusione con un amplificatore transbridge. Regolare la pressione di perfusione basale a 0 mmHg quando il cuore non è collegato al sistema di perfusione. </li><li> Allineare le telecamere di mappatura ottica per garantire l&#39;allineamento spaziale tra il V m e SR Ca 2 + segnali. <ol><li> In primo luogo, mettere a fuoco le telecamere posizionando un sovrano o di un oggetto con il testo nel piatto perfusione. Girare le telecamere di mapping in una modalità di visualizzazione dal vivo e regolare la messa a fuoco fino a quando il testo è nitido e chiaro. Acquisire un&#39;immagine singolo fotogramma da ogni telecamera. </li><li> Sovrapposizionequeste immagini e regolare la trasparenza dell&#39;immagine superiore in modo che entrambe le immagini sono visibili. Il testo delle immagini dovrebbe sovrapporsi esattamente. Se non perfettamente allineati, regolare l&#39;angolo dello specchio dicroico o la posizione di ciascuna telecamera fino a quando le due immagini vengono allineate. </li></ol></li></ol> 2. la raccolta, Perfusione, e Dye-caricamento di Rabbit Heart <ol><li> Fissare il coniglio in un dispositivo di immobilizzazione di coniglio approvato. Profondamente anestetizzare tramite iniezione endovenosa (vena marginale dell&#39;orecchio) di sodio pentobarbital (50 mg / kg) ed eparina (1000 IU). <ol><li> Quando viene visualizzato il coniglio mancanza di riflessi nocicettivi, fare una incisione cutanea mediana per esporre lo sterno e costole. Tagliare lo sterno con blunt punta forbici chirurgiche dal xifoidea al manubrio. Fare attenzione a non danneggiare il cuore durante l&#39;apertura dello sterno. Stendere le costole per esporre il cuore. </li><li> Accise rapidamente l&#39;intero blocco cuore-polmone per rapidamente tagliando tutte le navi e tessuto connettivo. Immediatamente posizionare il blocco cuore-polmone in soluzione Tyrode ghiacciata. </li></ol></li><li> Individuare l&#39;aorta per perfusione retrograda. Tagliare l&#39;aorta ascendente prossimale solo per i tre rami dell&#39;arco aortico. Incannulare l&#39;aorta di una cannula 8 G connesso al sistema di perfusione. Usare un pezzo di USP sutura di seta 0 per fissare l&#39;aorta sulla cannula. </li><li> Sezionare con cura la trachea, polmoni, e il grasso epicardico dal cuore. Con una pinza taglienti e forbici dissezione, localizzare la valvola mitrale e accuratamente danneggiare o rimuovere un foglio per prevenire la congestione soluzione nel ventricolo sinistro. </li><li> Immergere il cuore nella camera di perfusione di vetro rivestito orizzontalmente con la superficie anteriore del cuore rivolto verso l&#39;alto per l&#39;imaging. Fissare la cannula di un pezzo di Sylgard con perni a forma di U sul fondo del piatto di silicio per impedire il movimento del cuore durante la mappatura (Figura 1D). Se lo si desidera, inserire un piccolo perno insetto all&#39;apice del cuore per STAbilization. </li><li> Posizione elettrocardiogramma (ECG) elettrodi nel bagno sul lato destro e sinistro del cuore. Immergere completamente gli elettrodi nel bagno e assicurarsi che non sono in contatto con la superficie del cuore di fornire un ECG volume analogo condotto ad una configurazione che piombo. Verifica di un lead normale che ECG morfologia. Regolare il flusso (25 - 35 ml / min) per garantire che la pressione aortica è mantenuta a 60 - 70 mmHg. </li><li> Spegnere le luci della stanza e aggiungere ,3-0,6 ml di soluzione madre blebbistatin (passo 1.2) al perfusato (concentrazione finale 10-20 micron). <ol><li> Spegnere luci della stanza per evitare photoinactivation di blebbistatin. Se necessario, una piccola riflettore o proiettore possono essere utilizzati per fornire l&#39;illuminazione di operazione. Nota: blebbistatin è un inibitore ATPasi miosina e un disaccoppiatore eccitazione-contrazione. Perché Fluo-5N di carico richiede estesi (60 min) RT (ipotermia) le condizioni (vedi i punti 2.8 - 2.10), la produzione di energia cardiaco può essere compromessa. Therefore, l&#39;aggiunta di blebbistatin nella prima perfusato per tingere di carico può ridurre la domanda di energia 16 ed eliminare gli artefatti da movimento durante le successive registrazioni ottiche 17. </li></ol></li><li> Quando la contrazione del cuore ha cessato (10 - 15 min, verificato sulla registrazione pressione aortica), passare al sistema di ricircolo perfusione piccolo volume (vedi punto 1.4.1). </li><li> Preparare e caricare la soluzione di carico Fluo-5N AM: Aggiungere 0,25 ml di Fluo-5N AM soluzione madre (passo 1,3) a 0,25 ml caldo del 20% F127 Pluronic. Aggiungere 0,5 ml di soluzione calda Tyrode al composto, mescolare bene e aggiungere al sistema di ricircolo perfusione piccoli volumi. </li><li> Monitorare continuamente pressione di perfusione e ECG e regolare il flusso, se necessario. Dye caricamento dura circa 1 ora a temperatura ambiente. 45 minuti dopo il caricamento è iniziato, accendere il bagno di acqua circolante per iniziare il riscaldamento del sistema di perfusione e perfusato a 37 ° C. </li><li> Dopo 60 min di Fluo-5N AM caricamento, SWItch la perfusione per il volume del sistema di ricircolo perfusione più grande (vedi punto 1.4). </li><li> Diluire 50 ml di soluzione colorante sensibile RH237 magazzino tensione (passo 1.3) in 1 ml di soluzione calda di Tyrode e aggiungere lentamente (sopra ~ 5 min) a una porta di iniezione prossimale alla cannula. </li></ol> 3. mappatura ottica <ol><li> Durante i momenti finali di colorante carico, posizionare il cuore e mettere a fuoco le telecamere di mappatura ottica per garantire campo appropriato della vista per l&#39;esperimento. </li><li> Se lo si desidera, posizionare un elettrodo di stimolazione bipolare sulla superficie epicardica del cuore per la stimolazione. </li><li> Posizionare una custodia di plastica o di vetro sulla superficie della camera di perfusione per ridurre artefatti da movimento sulla superficie del liquido che può essere presente a causa di ricircolo del perfusato. Nota: In alternativa un vetrino, utilizzare una plastica di copertura pulito 50 millimetri Petri piatto. </li><li> Focalizzare le guide di luce per illuminare uniformemente la superficie del cuore con eluce xcitation. Utilizzare diodi emettitori di luce blu (LED) fonti di luce e filtrare ulteriormente la luce con un 475-495 filtro passa-banda nm (Figura 1A). </li><li> Raccogliere fluorescenza emessa con un macroscope e due complementari metallo-ossido-semiconduttore (CMOS) telecamere mappatura ottica. Dividere la luce emessa con uno specchio dicroico a 545 nm. <ol><li> -Pass filtro lungo la lunghezza d&#39;onda più lunga porzione, contenente il segnale V m, a 700 nm. Filtro passa-banda della lunghezza d&#39;onda più corta porzione, contenente il segnale SR Ca 2+, da 502 - 534 nm (Figura 1A). Impostare la frequenza di acquisizione dati ottici a 0,5 - 1 kHz. </li></ol></li><li> Per ogni registrazione ottica, prima di avviare il protocollo di stimolazione desiderata, accendere la luce di eccitazione, e raccogliere 1-4 sec di dati. Se lo si desidera, sincronizzare i protocolli di stimolazione e acquisizione dati. Nota: Non è possibile ricaricare il Fluo-5N AM, quindi il rapporto segnale-rumore (SNR) si decrease tutto l&#39;esperimento a causa di perdite dal tingere SR. Limitare protocollo sperimentale per 1-2 ore per garantire elevati valori di SNR. </li><li> Dopo l&#39;esperimento, lavare il sistema di perfusione nella sequenza di acqua deionizzata, 70% alcool reagente, e di nuovo con acqua deionizzata. </li><li> Filtro ogni insieme di dati con un filtro gaussiano spaziale (raggio 3 pixel) utilizzando i pacchetti software disponibili in commercio secondo il protocollo del produttore. </li><li> Se necessario, spazialmente allineare il V m e SR Ca 2+ insiemi di dati. Come al punto 1.6, sovrapporre le immagini da ogni telecamera e verificare che le strutture anatomiche del cuore (es., Vascolare coronarico, bordi di atri o ventricoli) o altri oggetti nel campo visivo (ad esempio, la cannula, elettrodi di stimolazione) esattamente sovrapposizione. In caso contrario, l&#39;allineamento spaziale delle serie di dati è necessario. </li><li> Spostare l&#39;immagine in alto trasparente si ottiene X e Y-direzione fino sovrapposizione esatta, prendendo nota del numero di pixell&#39;immagine deve essere spostata in ciascuna direzione. L&#39;intero insieme di dati corrispondente all&#39;immagine superiore (o V mo SR Ca 2+) deve quindi essere spostata dal numero determinato di pixel in ogni direzione per assicurare un allineamento preciso tra V m e SR Ca 2+ set di dati. </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.

Le chiavi del successo Fluo-5N colorante di caricamento sono configurazione ricircolo perfusione piccolo volume, che consente una elevata concentrazione Fluo-5N, senza la necessità di grandi quantità di colorante, la lunghezza del tempo di caricamento (1 ora), ed eseguire il caricamento a RT. Se il caricamento viene eseguito a temperature fisiologiche, attività enzimatica cellulare rapidamente scinde il tag -AM quando il colorante attraversa la membrana cellulare, intrappolando le molecole di colorante nel citosol e ...

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 figura 1A mostra un diagramma schematico della configurazione ottica per la doppia V m e SR Ca 2+ mappatura. Con questa configurazione, vi è la completa separazione spettrale del Vm e SR Ca 2 + segnali (Figura 1B). Lo schema del sistema di perfusione a doppio loop usato per Fluo-5N colorante carico è illustrato nella Figura 1C. Figura 1D mostra l&#39;orientamento orizzontale del cuore nel piatto perfusion...

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

Questo articolo viene descritto il protocollo dettagliato e le attrezzature necessarie per la mappatura ottica doppio di transmembrana potenziale (V m) e senza reticolo intra-sarcoplasmatico (SR) Ca 2+ nel cuore di coniglio Langendorff-perfusione. Questo metodo consente l&#39;osservazione diretta e la quantificazione delle Vm e SR Ca 2+ dinamica nel cuore intatto.

Mappatura ottica di Intra-reticolo sarcoplasmatico Ca<sup&gt; 2+</sup&gt; E transmembrana potenziale nel Rabbit Heart Langendorff-perfuso

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 Views.  University of California Davis.  Questo articolo viene descritto il protocollo dettagliato e le attrezzature necessarie per la mappatura ottica doppio di transmembrana potenziale (V m) e senza reticolo intra-sarcoplasmatico (SR) Ca 2+ nel cuore di coniglio Langendorff-perfusione. Questo metodo consente l'osservazione diretta e la quantificazione delle Vm e SR Ca 2+ dinamica nel cuore intatto. 

Video: 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.

Manutenzione e pulizia adeguata del microscopio

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

Ricerca

Biologia

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).

Mappatura ottica di Langendorff-perfusi Hearts Rat

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.

Visualizzare il cuore pulsante di Drosophila</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.

Semiautomatici Analisi Heartbeat ottica di piccoli cuori

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.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

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.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

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

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.

Valutazione della distribuzione spaziale delle γH2AX seguenti radiazioni ionizzanti

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.

Clonogenica saggio: cellule aderenti

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.

Tensione e Calcio mappatura ottica a doppio canale di Colta HL-1 atriale miociti monostrato

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.