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

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

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Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells

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1. Paralell recording of voltage and calcium signals

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

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

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

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

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

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

Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells

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4-[2-[6-(Dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium

Electrochromic Photo-induced electron transfer

2. FluoVolt Dye

Di-4-ANEPPS

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

Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells

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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

Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells

2. FluoVolt Dye

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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

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)

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)

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)

Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells

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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

Experimental parameters for an efficient recording of membrane potential and calcium activity from hIPS-derived cardiomyocytes in 96 wells

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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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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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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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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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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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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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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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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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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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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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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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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