Intracellular Calcium Imaging of Cardiac Cells

Ca²⁺ imaging was performed according to a previously published protocol^{69 (link)}. Briefly, cells were loaded with 5 mM of fluo-4 fluorescent Ca²⁺ indicator (Molecular Probes) in the presence of Pluronic F-127 (Molecular Probes) to allow the recording of intracellular Ca²⁺-transients. For pacing, scaffolds were plated on a 35-mm optical plate (Matek) with field stimulation electrodes (RC-37FS; Warner Instruments) and paced using a stimulus isolation unit (SIU-102; Warner Instruments), by applying 5 ms-suprathreshold bipolar stimulation pulses up to 50 mA. Intracellular Ca²⁺-transients were recorded using a Zeiss laser-scanning confocal imaging system (Fluo-view; Olympus) mounted on an upright BX51WI Olympus microscope equipped with a X60 water objective. Data were analyzed utilizing a MatLab-based custom-written software.

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Efraim Y., Schoen B., Zahran S., Davidov T., Vasilyev G., Baruch L., Zussman E, & Machluf M. (2019). 3D Structure and Processing Methods Direct the Biological Attributes of ECM-Based Cardiac Scaffolds. Scientific Reports, 9, 5578.

Publication 2019

Pluronic F-127 RC-37FS SIU-102 BX51WI Olympus microscope

Cells Fluo Fluo 4 Intracellular Isolation Microscope Molecular probes Pluronic f 127 Pulses Transients

Corresponding Organization :

Other organizations : Technion – Israel Institute of Technology

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Variable analysis

independent variables

Pacing of scaffolds using field stimulation electrodes by applying 5 ms-suprathreshold bipolar stimulation pulses up to 50 mA

dependent variables

Intracellular Ca2+-transients

control variables

Cells were loaded with 5 mM of fluo-4 fluorescent Ca2+ indicator in the presence of Pluronic F-127 to allow the recording of intracellular Ca2+-transients
Intracellular Ca2+-transients were recorded using a Zeiss laser-scanning confocal imaging system mounted on an upright BX51WI Olympus microscope equipped with a X60 water objective

controls

No positive or negative controls were explicitly mentioned in the protocol

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