| In Vitro | In vitro activity: Flupirtine pre-incubated for 2 hours prevents cell death in rat cortical neurons induced by NMDA and gp120 of HIV-1. Flupirtine is capable of protecting primary neurons against glutamate-induced cytotoxicity by reducing calcium ion concentrations at 1-10 mM. Flupirtine pretreated for 2 hours preventsβ-amyloid-induced apoptosis in primary neuronal cells at concentrations of 1 or 5μg/mL.
Cell Assay: For measurement of viability and generation of reactive oxygen intermediates, PC12 cells are seeded in 24- or 96-well plates coated with poly-L-lysine at 105 cells/mL. Drugs are dissolved in PBS (pH 7.4), or ethanol and filtered sterile. At the end of each experiment cells are trypsinized and pelleted together with cells of the culture supernatant. After staining for 10 min with 0.2% Trypan blue solution live (unstained) and dead (Trypan blue positive) cells are counted in a hemocytometer chamber. In addition, cellular viability is evaluated by the reduction of MTT to formazan. After 2 hours incubation with MTT (0.5 mg/ml) at 37 °C, cells are lysed in DMSO. Extinction at 570 nm is determined on a plate photometer. For staining of surviving adherent cells, plates are incubated for 10 min with 0.5% crystalviolet dissolved in 20% methanol. Plates are rinsed with water and stained cells are lysed in 50% ethanol, 0.1 M sodiumcitrate before determining extinction at 550 nm. |
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