Tcs sp5 mp inverted microscope

Cells were loaded with Fluo-3 AM (4 μM, Life Technologies, Foster City, CA, USA) using conventional protocols as recommended by the manufacturer. In brief, neuronal cultures were incubated with Fluo-3 AM for 45 min in the dark at room temperature. Then, Fluo-3 AM was washed out, and cells were incubated in the external solution for another 30 min in the dark. Coverslips with Fluo-3-loaded cultures were placed in the perfusion chamber, which was mounted on the stage of a Leica TCS SP5 MP inverted microscope (Leica Microsystems, Germany). Fluorescence was activated with 488 nm laser light and emission was measured within the wavelength range from 500 to 560 nm. Images were captured every minute during 60 min experiments. In Ca²⁺ imaging experiments to activated NMDARs 100 μM DL-HCY and 30 μM Gly were used.

Cells were loaded with 10 μM Fluo-3 AM (Life Technologies, CA, USA) using conventional protocols as recommended by the manufacturer. In brief, hMESCs were incubated with Fluo-3 AM for 60 min in Ca²⁺-free basic solution (144 mM NaCl, 4 mM KCl, 0.33 mM NaH₂PO₄, 5.5 mM glucose, 0.53 mM MgCl₂, 10 mM HEPES (Sigma-Aldrich, MO, USA), pH adjusted to 7.4 using NaOH) in the dark at room temperature. Then, Fluo-3 AM was washed out, and cells were incubated in the external basic solution with 4 mM CaCl₂ (Sigma-Aldrich, MO, USA) for another 15 min in the dark. Coverslips with Fluo-3-loaded cultures were placed in the perfusion chamber, which was mounted on the stage of Leica TCS SP5 MP inverted microscope (Leica Microsystems, Germany). Fluorescence was activated with 488 nm laser light and emission was measured within the wavelength range from 500 to 560 nm. Images were captured every 2.5 seconds during ∼ 60 min experiments.
In Ca²⁺ imaging experiments we used basic solution containing 144 mM NaCl, 4 mM KCl, 0.33 mM NaH₂PO₄, 5.5 mM glucose, 4 mM CaCl₂, 0.53 mM MgCl₂, 10 mM HEPES, pH adjusted to 7.4 using NaOH. In Ca²⁺-free experiments we used the same basic solution without CaCl₂. In order to avoid Ca²⁺ contamination in Ca²⁺-free experiments, basic solution was additionally supplemented with 4 mM EGTA (Sigma-Aldrich, MO, USA).

Cortical neurons were incubated within the basic solution containing 5 µM rhodamine-123 for 30 min. The dye fluorescence was detected using a Leica TCS SP5 MP inverted microscope (Leica Microsystems Inc.). The wavelengths of 488 nm and 510–530 nm were used for excitation and emission, respectively, to monitor mitochondrial inner membrane potential (φ_mit). The sampling interval was set to ~1 s (frame 512 × 512 px). In experiments, HCY (100 µM) or glutamate (100 µM) were always co-applied with 30 µM glycine. To test the functional state of mitochondria, the oxidative phosphorylation inhibitor, 4 µM CCCP (m-chlorophenyl hydrazone, Sigma, St. Louis, MO, USA) was added at the end of experiment [43 (link)].