SEA0400 is a selective inhibitor of the Na+/Ca2+ exchanger.

NCX:5 -33 nM.

Na+-Ca2+ exchange activity is determined by assaying Na+-dependent 45Ca2+ uptake as reported previously. Briefly, the cells are preincubated in Hanks' balanced saline solution (HBSS) for 20 min, and the medium is switched to HBSS containing 45Ca2+?and incubated for 5 min. To increase intracellular Na+?concentration, 1 mM ouabain plus 20 μM monensin (for astrocytes and microglia) and 10 μM monensin (for neurons) are used. Monensin is added simultaneously with the isotope. Ouabain is added 5 min before monensin in astrocytes and microglia. SEA0400 and KB-R7943 are added 5 min before monensin and present during 45Ca2+?uptake reaction.

SEA0400 is dissolved in DMSO (final concentration 0.1%). Cells, plated in 96-well plastic tissue culture plates, are incubated at 37°C for 30 min in normal or Ca2+-free HBSS containing 10 μM H2DCF-DA and 0.25 μg/mL Cremophor EL, and then rinsed twice with normal HBSS to remove excess dye. The cells are reperfused in normal HBSS for 1 h, and the conversion of H2DCF-DA to its fluorescent product dichlorofluorescein by ROS, presumably Water2 and hydroxyl radical, is determined with excitation at 485 nm and emission at 535 nm using a Wallac Multilabel counter. ROS production is expressed as a percentage of control cells. The linearity and sensitivity of ROS assay are confirmed using Water2 prior to the experiment. SEA0400 at the indicated concentrations is added 10 min before Ca2+?reperfusion and present until assay.

SEA-0400, SEA 0400, 2-[4-[(2,5-二氟苯基)甲氧基]苯氧基]-5-乙氧基苯胺

DMSO: 50 mg/mL (134.63 mM), Sonication is recommended.

10% DMSO+90% Corn Oil: 2.5 mg/mL (6.73 mM), Sonication is recommended.