Quantifying Mitochondrial Network Morphology

Cells were adhered to chamber slides at 2x10³/well and cultured with or without 1ng/ml TNFα for 24 hours. Slides were fixed in acetone, air-dried and stored at -20°C. Slides were rehydrated in PBS, blocked in 10% normal goat serum and incubated with mouse anti-TOMM20 (4F3, Abcam, UK) prior to goat anti-mouse IgG1 (Southern Biotech, Birmingham, USA). The FITC signal was amplified with anti-FITC Alexa Fluor 488 (Life Technologies) and nuclei were stained with Hoechst 33258. Slides were mounted in Prolong Diamond (Life Technologies) before imaging. Images were captured on the Leica DM6000 using the proprietary software and processed using Fiji (34 (link)) in a method adapted from (35 (link)). In brief, the image in the TOMM-20 channel was sharpened, thresholded, converted to a mask and then skeletonized prior to running the binary connectivity plug-in as described (36 (link), 37 (link)). For visualisation the Glasbey lookup table was used and numbers of each pixel connection type were exported. Nuclei were counted as an assessment of cell number in each field of view and these data were combined.