ROS estimation

The ROS levels were measured in astroglial cultures exposed to ALS-CSF using dichlorofluorescin diacetate (DCFDA), a compound that reacts with the intracellular ROS to produce fluorescence. Briefly, the cultures were treated with DCFDA, harvested, and lysed, and the resultant fluorescence release was measured at 536 nm in an ELISA plate reader. Fluorescence/cell was calculated and statistically analyzed. For qualitative studies, the cells were plated onto 13-mm coverslips. After exposure, the cells were incubated with Locke’s solution and DCFDA and directly viewed (excitation—480 nm; emission—530 nm) using confocal microscopy (Leica TCS-SL, Leica Microsystems, Germany).