4.9. Intracellular Calcium Measurement

Scanning laser confocal microscopy (Leica TCS SP2, Wetzlar, Germany) was used to determine cytosolic Ca²⁺ in primary cultured hippocampal neurons at day in vitro 12 (DIV12). The cells were loaded with 5 μM Fluo-4/AM (Beyotime, Shanghai, China) in Hank’s solution at 37 °C in the dark for 45 min, then washed using Hank’s solution gently to remove extracellular Fluo-4/AM dye. Fluorescence was monitored immediately every 10 s over a period of 15 min (excitation at 488 nm and emission at 526 nm). Prior to NMDA stimulation, the dye-loaded cells were scanned for ~2 min to obtain a basal level of fluorescence intensity. After different cell treatments as mentioned above, the fluorescence intensity was monitored and the dynamic change of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was recorded. The [Ca²⁺]_i value was measured according to the relative fluorescence intensity to the basal level.