Lsm axio observer z1 confocal microscope

Intracellular calcium (Ca²⁺) transients were imaged in iPSC-CMs at days 70–90 of cardiac differentiation as described [51] (link). Briefly, 250,000 iPSC-CMs were plated onto Geltrex-coated 25-mm round coverslips. After 5–7 days, iPSC-CMs were transduced with AAV6 viruses. For imaging, iPSC-CMs were incubated with 2.5 µM of a Ca²⁺‐fluorescent probe RHOD-2 (Catalog No. R1244, Invitrogen) for 30 min in Tyrode’s solution containing 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose, 1.8 mM CaCl₂, and 10 mM HEPES (pH 7.4). iPSC-CMs were paced at 0.25 Hz and Ca²⁺ transients were recorded at room temperature with a Zeiss LSM Axio Observer Z1 confocal microscope with the following settings: Laser 516 nm with 0.4%–1.8 %, line scan mode with 20,000 cycles and zoom 3. For statistical analysis, the fold change was calculated by dividing the SLM2 overexpression by the mean of the basal mock control values.

For the analysis of the Ca²⁺ kinetics, cytosolic calcium (Ca²⁺) transients were imaged using Fluo-4 probe (Invitrogen). For this purpose, 300.000 iPSC-CMs were plated onto Geltrex-coated 25 mm round coverslips. After 7–9 days, the iPSC-CMs were incubated with 2.5 µM of Ca²⁺-sensitive fluorescent probe Fluo-4 for 30 min in Tyrode’s solution containing 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, 1.8 mM CaCl₂ and 10 mM HEPES (pH 7.4). The iPSC-CMs were paced at 0.25 Hz, and Ca²⁺ transients were recorded at room temperature using a Zeiss LSM Axio Observer Z1 confocal microscope with the following settings: a laser of 488 nm with 0.4–1.8%, a line scan mode with 20,000 cycles and zoom factor of 3.