Live cell uorescence microscope

In order to identify potential of neuronal differentiation, we evaluated calcium in ux which is an indicator for neurotransmitter transmission. The intracellular calcium assessment was described in previous study (23) . The specimens were incubated with 3 µM Fluo-3 AM (Invitrogen) and 0.08% pluronic acid (Invitrogen) in DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin at 37 o C for 60 minutes. Subsequently, the specimens were washed with DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin, and PBS. The specimens were maintained in Tyrode's solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 30 mM glucose, and 25 mM HEPES, pH 7.4) (All from Sigma-Aldrich). The neurotransmitter releasing ability of differentiated cells was simulated with 50 mM KCI. The intensity of calcium was recorded time-lapse at excitation 506 nm for 3 minutes by the Live-cell uorescence microscope, IX83XDC (Olympus), and interpreted using the ImageJ program (NIH).

In order to identify potential of neuronal differentiation, we evaluated calcium in ux which is an indicator for neurotransmitter transmission. The intracellular calcium assessment was described in previous study (23) . The specimens were incubated with 3 µM Fluo-3 AM (Invitrogen) and 0.08% pluronic acid (Invitrogen) in DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin at 37 o C for 60 minutes. Subsequently, the specimens were washed with DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin, and PBS. The specimens were maintained in Tyrode's solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 30 mM glucose, and 25 mM HEPES, pH 7.4) (All from Sigma-Aldrich). The action potential ability of differentiated cells was simulated with 50 mM KCI. The intensity of calcium was recorded time-lapse at excitation 506 nm for 3 minutes by the Live-cell uorescence microscope, IX83XDC (Olympus), and interpreted using the ImageJ program (NIH).