Mitoid

H9C2 cells were fixed with 4% PFA, permeabilized with 0.5% Triton, blocked with 3% BSA and incubated with anti‐vinculin (Sigma) or anti‐pH2A.X (Millipore) overnight at 4°C. The secondary antibody was Alexa‐Green‐488 goat anti‐mouse (Invitrogen). Images were acquired using epifluorescence microscopy (DM600 microscope; Leica). For mitophagy detection, EGFP‐LC3 plasmid and MitoID (Enzo) were used to stain autophagosomes and mitochondria, respectively. Image acquisition was performed with an LSM780 laser scanning confocal microscope (Carl Zeiss). For mitochondrial 8‐OH‐dG detection, cells were fixed with methanol for 30 min at −20°C, permeabilized with 0.2% Triton and treated with RNase A at 37°C for 1 hr, followed by denaturation with ice‐cold 25 mM NaOH in 50% ethanol, as previously described (Ohno, Oka, & Nakabeppu, 2009). After blocking with 10% BSA, the cells were incubated with mouse anti‐8‐OH‐dG (clone 483.15; Millipore) overnight at 4°C. The secondary antibody was goat anti‐mouse Alexa 488.

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.

MITO-ID (Enzo Biochem, New York, NY), a live cell dye for actively respiring mitochondria was used to assess mitochondrial membrane potential and MitoTracker Red (Invitrogen, Waltham, MA), a live cell mitochondrial membrane-dependent dye, was used to image mitochondria (Figure 4). Following LPS treatment (see Table S1), microglia were incubated for 30 min with MITO-ID as per kit instructions. Then microglia were imaged immediately using the microplate reader followed by an additional 30 min incubation at 37°C then imaged a second time. Following LPS treatment (see Table S1), microglia were incubated for 10 min with 100 nM MitoTracker Red, then cells were fixed. Microglia were imaged using Zeiss confocal microscope and analyzed using Mito-Morphology Macro for FIJI as previously described (Wiemerslage and Lee, 2016 (link)). Briefly, MITO-ID images were uploaded into FIJI and the Mito-Morphology Macro was used to analyze the image for mitochondrial characteristics. Both the MITO-ID and MitoTracker data are initially presented as raw data to compare the dPBS response data between WT and Fmr1 KO microglia. To clearly demonstrate the LPS-induced response, the data are then presented as normalized to the dPBS response for each genotype.