CM SR Ca2+ load and RyR2-mediated leakage were assayed using Fluo-4 fluorescence. Patient and unrelated control iPSC-CMs were dissociated from 24-well plates and then plated and cultured on Geltrex-coated (Gibco, Thermo Fisher Scientific, A1413202), 35 mm, glass-bottom dishes (MatTek, P35G-1.5-10-C) at 37°C, 5% CO2, for 3–5 days. CMs were incubated for 30 minutes in DMEM/2% FBS media containing 2 μM of Fluo-4 AM (Thermo Fisher Scientific, F14201) and 0.02% F-127 (Thermo Fisher Scientific, P3000MP). The cells were washed once with fresh DMEM/2% FBS media. During imaging, the dishes were kept in a heated 37°C stage-top environment chamber supplied with 5% CO2. Imaging of Ca2+ transients was taken under ×40 objective using Nikon Eclipse Ti light microscope. Ca2+ transients from single beating iPSC-CMs, paced at 0.25 Hz, were recorded at a speed of 20 ms per frame for 20 seconds at 15% LED power. After finishing baseline recording, appropriate amounts of ISO (MilliporeSigma, I2760) and caffeine (MilliporeSigma, C0750) were added into the recording dish dropwise. The raw data were exported to Excel software (Microsoft) and then analyzed with an in-lab–developed Excel-based program.
I2760
Manufactured by Merck Group
The I2760 is a lab equipment product manufactured by Merck Group. It is a versatile instrument designed for precise measurement and analysis tasks within a laboratory setting. The core function of the I2760 is to provide accurate and reliable data collection and processing capabilities to support various scientific research and development activities. For detailed information on the specific features and intended applications of this product, please consult the official product documentation or contact our sales representatives.
Automatically generated - may contain errors
Lab products found in correlation
Fluo 4 am, by Thermo Fisher Scientific (4 mentions)
F 127, by Thermo Fisher Scientific (2 mentions)
Geltrex coated, by Thermo Fisher Scientific (1 mentions)
C0750, by Merck Group (1 mentions)
P35g 1.5 10 c, by MatTek (1 mentions)
Tyrode s solution, by Thermo Fisher Scientific (1 mentions)
Eclipse ti, by Nikon (1 mentions)
35 mm glass bottom dishes, by MatTek (1 mentions)
Myopacer field stimulator, by IonOptix (1 mentions)
2 protocols using i2760
1
iPSC-CM Ca2+ Transient Analysis
2
Calcium Imaging of Induced Pluripotent Stem Cell-Derived Cardiomyocytes
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iPSC-CMs cultured on 35-mm glass-bottom dishes (MatTek Corporation, Ashland, MA, USA) at 37°C, 5% CO2 were loaded with 2 μM Fluo-4 AM (Thermo Fisher Scientific, Waltham, MA, USA) with 0.02% F-127 (Thermo Fisher Scientific, Waltham, MA, USA) in Tyrode’s Solution (Alfa Aesar, Tewksbury, MA, USA) for 30 min. Following washout, Tyrode’s Solution was added, and cells were imaged. During imaging, cells were kept in a heated 37°C stage-top environment chamber supplied with 5% CO2. Imaging of Ca2+ transients was taken under a 40× water objective using a Nikon Eclipse Ti (Melville, NY, USA) light microscope. iPSC-CMs were paced at 0.5 Hz using an IonOptix MyoPacer Field Stimulator (Westwood, MA, USA). Time-lapse videos were taken at a speed of 20 ms/frame for 20 s. For recordings with ISO, 1 μM ISO (I2760; MilliporeSigma) was added to the Tyrode’s Solution, and video recordings were taken between 6 and 10 min of incubation. The raw data were exported to Excel software (Microsoft, Redmond, WA, USA) and analyzed with a custom Excel-based script to correct for photo bleaching.
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