Analysis of NCX1 activity

H9c2-NCX1 cells, cultured on 25 mm coverslip, were loaded with 4 µM Fluo-4/AM for 30 min in the dark at room temperature (Molecular Probe, Eugene, OR), in a standard solution containing (in mM): NaCl 140, KCl 5, MgCl₂ 1, CaCl₂ 2, glucose 10, HEPES 20, pH 7.4 adjusted with NaOH. At the end of the Fluo-4/AM loading period, cells were washed and left in the standard solution for further 10 min to allow the complete de-esterification of the dye. Then the coverslips were placed into a perfusion chamber mounted onto the stage of an inverted Zeiss Axiovert 200 microscope. NCX1 activity was evaluated as Ca²⁺ uptake through the reverse mode by switching the standard solution to a Na⁺-free solution containing (in mM): LiCl 140, KCl 5, MgCl₂ 1, CaCl₂ 2, glucose 10, HEPES 20, pH 7.4 adjusted with LiOH. [Ca²⁺]_i was measured by single-cell computer-assisted videoimaging using a LSM 510 confocal system (Carl Zeiss). Cells were treated according to the protocol schemes reported in Fig. 1. In particular, cells were exposed to glutamate and/or transporter inhibitors only during the reoxygenation phase or, for sham-treated cells (not subjected to hypoxia), under normoxia for equivalent length of time, and were not included in solutions used for Fluo-4/AM loading or fluorescence monitoring. Excitation light was provided by an argon laser at 488 nm and the emission was time-lapse recorded at 505-530 nm. Images were acquired every 5 s. Analysis of fluorescence intensity was performed off-line after images acquisition, as described before^17,20.