To measure phospholipidosis (PLD), LipidTOX Red phospholipidosis detection reagent was used according to the instructions for HCS LipidTOX Phospholipidosis and Steatosis kit (ThermoFisher Scientific). Briefly, LipidTOX Red reagent was warmed up to 37 C, diluted in the culture medium and filtered through 0.8 mm pore filter and added to cells together with test compounds. After 72 h incubation, cells were stained with 20 mg/mL Hoechst 33342 in 4% paraformaldehyde solution in PBS for 30 min, washed in PBS 3 times, and imaged on ImageXpress Micro (Molecular Devices) high content analysis system. LipidTOX Red was imaged in the Texas Red channel (Ex 562/40, Em 624/40) and Hoechst in the DAPI channel (Ex 377/50, Em 447/60). Four images were taken per well using a 10Â objective.
Alternatively, in some experiments, PLD was measured by staining the cells with NBD-phosphatidylethanolamine (NBD-PE) (Molecular Probes, ThermoFisher Scientific) during 72-h incubation with test compounds (referred to as PLD NBD-PE) as described earlier. 19
Alternatively, in some experiments, PLD was measured by staining the cells with NBD-phosphatidylethanolamine (NBD-PE) (Molecular Probes, ThermoFisher Scientific) during 72-h incubation with test compounds (referred to as PLD NBD-PE) as described earlier. 19