Lyso id

The fluorescent properties of IB were utilized for localization analyses. IB is excited at 568 and emits fluorescence at >650. ER-tracker Green was purchased from Thermo-Fisher (E34250) and Golgi-ID and Lyso-ID were purchased from Enzo (ENZ-51028-K100 and ENZ-51034-0100). These dyes were chosen because they have no spectral overlap with IB. Hoechst stain provided in the Enzo kits was used for nuclear counterstain. Staining was performed according to manufacturer’s protocols. Cells were concurrently loaded with 150 nM IB. High resolution live-cell imaging was performed on the DeltaVision OMX (GE Healthcare). Post-acquisition data analysis was done using Fiji software (Fiji.sc).

Cells were plated at 700000 cells/T25 flask and grown for 12 days before exposure to CM for 24 h. Cells were detached using PBS EDTA and accutase. For flow cytometry there were 25000 cells per sample and for Image stream analysis there were 50000 cells per sample. Antibodies were used for the detection of astrocytes (GFAP 1:500 Millipore or GLAST 1:50 eBioscience), neurons (MAP2 1:2000 Synaptic systems 188 004 or CD90 1:200 eBioscience), autophagy (LC3 1:200 MBL and LysoID 1:500 Enzo Life Science) and a live/dead marker (1:2000, L23105 Life Technologies) and cell death (Annexin V 1:50, Life Technologies L23105). Mitochondrial function was accessed using Mitotracker Green FM 150 nM M7514, NaO 100 nM A1372, Mitosox 5 μM M36008 (all available from Life Technologies), and MitoID 1:2500 ENZ-51018, (Enzo Life Sciences). Cells were labelled for cell markers and mitochondrial functional indicators and the level of NaO, mitochondrial membrane potential, mitochondrial oxidative stress, and mitochondrial number. Cells from each exposure were compared for statistical difference from control. Flow cytometry was carried out on the BD LSRII and analysed using FloJo v 8.8.6. Cells prepared for detection of autophagy were run through Imagestream and analysed using IDEAS 6.1.303 software.