Staurosporine

Cell death was analyzed in astrocytes at 3 days post plating after O/N incubation with DMSO (solvent control) or 0.5 μM Staurosporine (Euromedex; LS9300-A). Cells were trypsinized, counted and 10⁶ cells were stained with a mix of 5% Annexin V-FITC (BD Horizon; 556547) to detect apoptotic cells, PI to detect necrotic cells and 2.5% Cd11b-V450 (BD Horizon; 560456) to exclude possible microglia contamination. Staining was performed for 15 min, prior to FACS analysis of 10,000 cells per embryo. Six different biological replicates were analyzed per genotype. The percentage of AnnexinV-positive, PI-positive WT and DMSXL astrocytes (negative for Cd11b staining) was analyzed in DMSO and Staurosporine conditions, using FlowJo software (BD Biosciences).

The following primary antibodies were used: anti-MHC (MF20, Developmental Studies Hybridoma Bank, 1:1,000), anti-active-caspase 3 (ab13847, Abcam, 1:500), anti-cleaved caspase 3 (9661, Cell Signaling, 1:500), anti-YFP (ab6673; Abcam, 1:1,000), anti-RFP (600-401-379, Rockland, 1:500), anti-myc (05-724; Millipore, 1:100), anti-P53 (sc-99, sc-6243, Santa Cruz, 1:200), anti-MDM2 (sc-965, Santa Cruz, 1:200), anti-P19 (ab80, abcam, 1:500) and anti-GAPDH (AM4300, Invitrogen, 1:1,000). Appropriate horseradish peroxidase-conjugated and Alexa Fluor-conjugated secondary antibodies were used for western blot and immunofluorescence studies. The following reagents were used: Staurosporine (BD Biosciences) was used in concentrations ranging between 0.005 and 1 μM. Myoseverin (Sigma) was used at 25 μM. 4,4′ diisothiocyanatostilbene-2,2′-disulphonic acid (Molecular Probes) was used at 100 μM. TMRE (Invitrogen) was used at 10 nM. Z-VAD (Santa Cruz) or Q-VD-OPH (Santa Cruz) was used at 10 μM. YO-PRO-1 (Molecular Probes) was used at 1 mM. FLIVO tracer was from ImmunoChemistry Technologies. TUNEL assay was performed with In Situ Cell Death Detection Kit (Roche). EdU staining was performed by incubating 30 min with 100 mM Tris, 1 mM CuSO₄, 10 μM fluorescent azide and 100 mM ascorbic acid43 (link).