I work with ipsc-CMs and we are trying to measure the action potential of the cells with Fluovolt. After preparation and incubation as described by the manufacturer, we were able to visualize the signal peak before the contraction. But after recording cells for a couple of minutes (1-2min), the frequency decreases along with fluorescence reduction until the cells stop completely and we can no longer see any signal (baseline fluorescence continues). We observed that this problem starts right after fluovolt excitation, as it does not occur in bright field (end of contraction).
A few notes> we add Blebbistatin (10uM) in the incubation solution but we already tested without it. We also tested 3 different dilutions of fluovolt and different light intensities and the problem remains.
Hello.
Fluovolt usually gives a fairly stable signal (at least in neonatal rat cardiomyocytes). A possible reason for the rapid decay of the signal may be the presence in the medium of fluorescence quenchers, toxic substances (for example, sodium azide), complexing agents (for example, EDTA), as well as violations of the ionic composition of the incubation solution (for example, there are no calcium and magnesium ions in the incubation solution). FluoVolt's photobleaching usually does not occur as so quickly.
Sincerely your,
Artem Trofimenko
Hi Artem Trofimenko, thanks for your advice. I will test on neonatal CM to see if the problem persists. We prepare the FV solution in Tyrode's and we don't add any substances other than those provided in the kit. The most bizarre thing is that we observe the problem only after fluorescence excitation. In bright field the CMs continue to contract.
With high settings of the excitation light (> 30–40% of Sola output) the dye had high phototoxicity and caused 100% cell death within minutes after a 30–60 s illumination (data not shown). Damaged cells “retaliated” by facilitating dye bleaching (possibly by releasing ROS) far beyond the bleaching rate in intact cells, the effect which could be misinterpreted for hyperpolarization. Fortunately, the phototoxicity was not noticeable at low light settings (< 10% of Sola output) and in practice less than 5% of the light output was adequate even for the fastest imaging (Pakhomov AG, Semenov I, Casciola M, Xiao S. Neuronal excitation and permeabilization by 200-ns pulsed electric field: An optical membrane potential study with FluoVolt dye. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2017 Jul 1;1859(7):1273-81).
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