Lysosomal membrane integrity

seed cells (SHSY-5Y) for 24:00:00 in a 12 well plate at a density of 1 x 10⁵ cells per well

incubate cells with 5 µg/ml acridine orange (disolved in media) for 0h 15m 0s at 37°C

discard medium and wash cells with 1mL of 1X PBS.

Add fresh medium to the wells.

treat cells with SPM (different concentrations: 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5 and 10 mM)) and positive control with chloroquine (500 µm) for 2h 0m 0s

at 37°C

collect cells and resuspend in 1X PBS + 1% BSA.

capture the mean fluoresence of 10 000 events with a flow cytometer.