hIPS-derived cardiomyocytes in 96 wells S. BEDUT For the Institut de - - PowerPoint PPT Presentation

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hips derived cardiomyocytes in 96 wells
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hIPS-derived cardiomyocytes in 96 wells S. BEDUT For the Institut de - - PowerPoint PPT Presentation

Dec 05, 2023 1.08k likes •1.44k views

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), Ple dExpertise Biotechnologie, Chimie &

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Experimental parameters for an efficient recording

  • f 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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SLIDE 2

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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SLIDE 3

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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SLIDE 4

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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SLIDE 5

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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SLIDE 6

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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SLIDE 7

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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SLIDE 8

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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SLIDE 9

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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SLIDE 10

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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SLIDE 11

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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SLIDE 12

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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SLIDE 13

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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SLIDE 15

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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SLIDE 16

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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SLIDE 17

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)

50 µl + 500 µl Powerload

d1, d2, d3, d4, d5 and d6

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SLIDE 18

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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SLIDE 19
  • 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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SLIDE 21
  • 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

  • rganic 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

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 2.0 500 1000 1500 2000 3000 4000

AMP

Time (hour)

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SLIDE 24
  • 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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SLIDE 26
  • 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 2.0 500 1000 1500 2000 3000 4000

AMP

Time (hour)

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SLIDE 27
  • 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 2.0 500 1000 1500 2000 3000 4000

AMP

Time (hour)

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SLIDE 28
  • 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