Cardiomyocytes voltage and calcium activity recording with a fast plate reader : 2. What are the basis of the signals recorded with FluoVolt?
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1 High throughput optical recording of voltage and calcium signals of human ips-derived cardiomyocytes for drug screening and cell lines characterization
2 Cardiomyocytes voltage and calcium activity recording with a fast plate reader : 1. How does it work? 2. What are the basis of the signals recorded with FluoVolt? 3. Is it accurate and how does it compares to MEA signals? 4. Why recording both voltage and calcium signals? 5. Exemples of application
3 FluoVolt : voltage-sensible fluorescent probe Fluorescein Close to : Oregon green (OGB1 & 2) Fluo-4 Cal-520 Photo-induced electron transfer modulation F/F % per 100 mv
4 FluoVolt : voltage-sensible fluorescent probe The FluoVolt signal follows efficiently the membrane potential variation FluoVolt signal Patch-clamp signal Photo-induced electron transfer modulation F/F % per 100 mv From McPheeters et al, PLOS ONE, aug. 24, 2017
5 FluoVolt Hamamatsu FDSS 7000EX plate reader 37 C High speed mode (16 ms sampling interval) Cal520
6 The fluorescent signal of a whole well is also representative of the local signal
7 The signal is a spatial and temporal summation of the cardiomyocytes monolayer action potentials in the well
8 All the wells are injected and recorded simultaneously Compound injection 1000 a.u.f. 500 a.u.f. 50 s
9 Why recording both voltage and calcium signals? The two signals don t allways vary in the same direction in response to drugs
10 Phenotypic studies of cell lines Nifedipine at 0.3 or 1 µm Acetylcholin at 1 µm Rising Slope of FV signal BPM
11 Phenotypic studies of cell lines Basal activity Drug 1 Drug 2 Drug 3 Drug 4 Drug 5 Drug 6 Drug 7 Drug 8 Total L L L L L L5 > L2 > L1 > L4 >> L3
12 Pluricytes No response to 1 µm Acetylcholine (ventricular-like response) 10 s
13 Pluricytes Injection of a herg positive compound EADs 10 s
14 Ready to use pre-seeded 96 well plate of Cor.4U (CardioPlate 96) FluoVolt (voltage signal) Cal520 (calcium signal)
15 Ready to use pre-seeded 96 well plate of Cor.4U (CardioPlate 96) FluoVolt
16 (transmembrane potential, Ionic currents) Patch-clamp Fluorescent voltage-sensitive dye - Transmembrane potential - Local or global indirect signal Extracellular (surface recording) Intracellular microelectrode - Transmembrane potential - True value - Local signal 0 mv MEA (Field potential recording) - Field potential recording - Local projected signal
17 MEA ECG «ECG-like» «T wave» 4 parameters : - Beat period (BPM) - Na spike amplitude - Na spike slope - Field potential duration (FPD)
18 MEA ECG «ECG-like» «T wave» 4 parameters : - Beat period (BPM) - Na spike amplitude - Na spike slope - Field potential duration (FPD) T wave morphologies
19 MEA FluoVolt/FDSS 4 parameters : - Beat period (BPM) - Na spike amplitude - Na spike slope - Field potential duration (FPD) 7 parameters for each dye : - Beat period (BPM) - Resting signal value (RMP) - Amplitude - Rising slope - Pulse width at 20 % (APD20) - Pulse width at 50 % (APD50) - Pulse width at 80 % (APD80)
20 MEA FluoVolt/FDSS + Acquisition rate (> 10 KHz) Label free Local recording (DADs) Long recordings possible - Few parameters Tricky interpretation Variability + Easy to operate Action potential related signal Throughput Reproducibility (averaged signal) - Frame rate (62 hz) Toxicity of the dyes
21 Phase 0 Phase 4 Phase 1-3
22 V C1 C2 C3 C4 C5 C6 C7 C8 C9 Semi-automatic analysis Early repolarization (D20) Late repolarization (D80)
23
24 H. MYSKOWSKI K. BRICE B. LOCKHART F. COGE N. VILLAIN Thank you for your attention
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