The astrocytes in primary culture were incubated for 10 min at 37°C with the MitoTracker® Red CMXRos probe (50 nM, Molecular Probes, USA). This probe passively allows diffusion across the plasma membrane and is accumulated in active mitochondria. Cells were then treated with an aldehyde-based fixative, 3.7% of paraformaldehyde to allow further sample processing. For the co-localization studies, the cells stained with MitoTracker were immunostained with NLRP3 and with caspase-1 activity labeling. For this end, cells were incubated for 10 min in PBS containing 0.1% Triton X-100, rinsed off with PBS (1×) and blocked for 1 h/RT with 10% normal goat serum in TBS/T (0.1%). After blocking, sections were incubated with the mouse monoclonal anti-NLRP3 (1/150, AdipoGEN) followed by Alexa fluor 405 (1/500, Molecular Probes). Then sections were incubated with caspase-1 fluorescence activity using the FAM-FLICATM Caspase-1 assay kit (ImmunoChemistry Technologies, USA). For this labeling, cells were incubated for 1 h at RT with the fluorescent FAM-YVAD-FMK FLICA reagent following the manufacturer’s instructions (ImmunoChemistry Technologies, USA). The reagent becomes covalently coupled to the active enzyme and is retained within the
