Experimental parameters for an efficient recording of membrane ...€¦ · 06/08/2016 ·...
Transcript of Experimental parameters for an efficient recording of membrane ...€¦ · 06/08/2016 ·...
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Experimental parameters for an efficient recording of membrane potential and calcium activity from
hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
For the Institut de Recherche Servier (IdRS), Pôle d’Expertise Biotechnologie, Chimie & Biologie, Croissy-sur-Seine, France
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
1. Parallel recording of voltage and calcium signals
2. FluoVolt dye
3. Cellular density (cells/well)
4. FluoVolt concentration
5. FluoVolt Toxicity
6. Washed-out calcium dyes
S. BEDUT
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
1. Paralell recording of voltage and calcium signals
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
1. Paralell recording of voltage and calcium signals
Phase 0 alteration
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
1. Paralell recording of voltage and calcium signals
Phase 1 and 3 alteration
Phase 4 alteration
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
1. Paralell recording of voltage and calcium signals
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
1. Paralell recording of voltage and calcium signals
![Page 8: Experimental parameters for an efficient recording of membrane ...€¦ · 06/08/2016 · Experimental parameters for an efficient recording of membrane potential and calcium activity](https://reader036.fdocuments.net/reader036/viewer/2022063008/5fbc1d76fd0bc97354411663/html5/thumbnails/8.jpg)
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
1. Paralell recording of voltage and calcium signals
![Page 9: Experimental parameters for an efficient recording of membrane ...€¦ · 06/08/2016 · Experimental parameters for an efficient recording of membrane potential and calcium activity](https://reader036.fdocuments.net/reader036/viewer/2022063008/5fbc1d76fd0bc97354411663/html5/thumbnails/9.jpg)
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
Electrochromic
F/F 6-10 % per 100 mV Rapid phototoxicity
Shift of the excitation/emission spectra
Photo-induced electron transfer
F/F 20-25 % per 100 mV ?
Modulation of the electron transfer rate
From Miller et al., PNAS 2012, 109 (6) : 2114-2119
2. FluoVolt Dye
Di-4-ANEPPS
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
4-[2-[6-(Dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium
Electrochromic Photo-induced electron transfer
2. FluoVolt Dye
Di-4-ANEPPS
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
2. FluoVolt Dye
4-[2-[6-(Dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium
Electrochromic Photo-induced electron transfer
Di-4-ANEPPS FluoVolt
Fluorescein
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
Di-4-ANEPPS
4-[2-[6-(Dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium
Electrochromic Photo-induced electron transfer
FluoVolt
2. FluoVolt Dye
Compatible with : Oregon green (OGB1 & 2) Fluo-4 Cal-520
Fluorescein
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
2. FluoVolt Dye
S. BEDUT
96 wells Experimental media: HBSS + 20 mM Hepes, pH 7.4 with NaOH (serum-free, phenol red-free) Temp: 37°C Loading time: 15 mn (50 µl, 37°C) Rinced twice (2 x 100 µl) Sampling interval: 16 ms
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
2500 5000 10 000 20 000 30 000 40 000 60 000
3. Cellular density (cells/well)
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
2500 5000 10 000 20 000 30 000 40 000 60 000
3. Cellular density (cells/well)
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
2500 5000 10 000 20 000 30 000 40 000 60 000
3. Cellular density (cells/well)
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
4. FluoVolt concentration
In 10, 20, 30, 40, 50 and 60 ml HBSS (d1 to d6)
0
50 µl + 500 µl Powerload
d1, d2, d3, d4, d5 and d6
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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
S. BEDUT
5. FluoVolt toxicity
In 10, 20, 30, 40, 50 and 60 ml (d1 to d6)
d1, d2, d3, d4, d5 and d6
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S. BEDUT
6. Washed-out calcium dyes
For an easy to operate assay, the same protocol as FluoVolt is expected
96 wells Experimental media: HBSS + 20 mM Hepes, pH 7.4 with NaOH Temp: 37°C Loading time: 15 mn (50 µl, 37°C) Rinced twice (2 x 100 µl) Sampling interval: 16 ms
Oregon green
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
Dye extrusion
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S. BEDUT
6. Washed-out calcium dyes
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
Start 30 mn
Fluo-4 (Molecular probes)
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S. BEDUT
6. Washed-out calcium dyes
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
OUT
IN
non polar, Ca2+ insensitive
esterases
polar, Ca2+ sensitive
X
FLUO-4
organic anions transporter
Probenecid
X
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S. BEDUT
6. Washed-out calcium dyes
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
0
200
400
600
800
1000
1200
Start 30 mn
Start
30 mn
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S. BEDUT
6. Washed-out calcium dyes
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
0.0 0.5 1.0 1.5 2.0 0
500
1000
1500
2000
3000
4000
AMP
Time (hour)
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S. BEDUT
6. Washed-out calcium dyes
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
vehicle dof. 20 nM dof. 20 nM +
0.5 mM Probe. dof. 20 nM +
0.75 mM Probe. dof. 20 nM + 1 mM Probe.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Amplitude BPM D80
dofetilide 20 nM
dof + 0.5 mM P.
dof + 1 mM P.
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S. BEDUT
6. Washed-out calcium dyes
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
Cal-520 (AAT bioquest)
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S. BEDUT
6. Washed-out calcium dyes
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
0.0 0.5 1.0 1.5 2.0 0
500
1000
1500
2000
3000
4000
AMP
Time (hour)
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S. BEDUT
6. Washed-out calcium dyes
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
0.0 0.5 1.0 1.5 2.0 0
500
1000
1500
2000
3000
4000
AMP
Time (hour)
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S. BEDUT
7. Conclusions
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
No serum, no phenol-red (HBSS + Hepes) No probenecid High cellular density 37°C 16 ms sample interval FluoVolt Cal-520 15 mn loading time 2 wash-outs About 3 hours of recording capacity
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S. BEDUT
Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells
H. MYSKOWSKI J.P. STEPHAN F. COGE V. LAMAMY N. VILLAIN
Thank you for your attention