Intracellular Calcium Dynamics in Neuronal Cells

αS species were added to the CM of SH-SY5Y cells and primary rat cortical neurons seeded on glass coverslips for 15 min at various concentrations (0.03, 0.1, 0.3, 1.0, and 3.0 μM). In a set of experiments, 0.3 µM OB* were added to the CM of SH-SY5Y cells for 0, 5, 10, 15, 30, 60, and 180 min and to primary rat cortical neurons for 0, 5, 15, 60, and 180 min. Cells were also treated with 0.3 µM OB* in CM without Ca²⁺. In a set of experiments, cells were treated for 15 or 180 min with 0.3 µM M in the absence or presence of 0.03 µM SF (monomers equivalents, corresponding to 10% of monomers). Then the cells were loaded with 10 μM Fluo-4 AM (Thermo Fisher Scientific) and the analysis was performed by confocal microscopy (excitation at 488 nm)^{37 (link),57 (link)}.
In a set of experiments, the Ca²⁺ influx was analyzed in real-time in living SH-SY5Y cells loaded with the Fluo-4 AM probe for 10 min. The intracellular Ca²⁺ basal level in living cells was measured for 10 min and then Ca²⁺ currents were analyzed following the addition of OB* or SF up to 30 min. The emitted fluorescence was detected at 488 nm excitation line over time by the confocal scanning system described above.