Figure 2
Mitochondrial function assay workflow and image analysis. Outline of assay workflow (A). iPSC-derived neurons stained with Hoechst (shown in blue), Calcein (shown in green) and TMRM (shown in red) (B), scale bar represents 50 μm. Nuclei identified from Hoechst staining were segmented using CellProfiler software. Each object is shown in a different arbitrary colour (C,i). Cell soma identified by Calcein staining were then segmented by propagation from each nuclei; each object is shown in a different arbitrary colour (C,ii). Hoechst-positive cells with no Calcein staining, assumed to be dead cells (arrows, Ci,ii) were removed from analysis (C,iii), leaving the final cells to be analysed, each shown in a different arbitrary colour (C,iii). For mitochondrial identification, the TMRM image (D,i) was enhanced with a white top-hat filter (D,ii), mitochondria were then segmented from the enhanced image, each shown in a different arbitrary colour (E,iii). Mitochondria were then associated with their related soma. For some measurements all mitochondria within a cell were classified as one object, shown as one colour for each associated cell (D,iv). Fluorescence intensity was measured from the original TMRM image, analysing either all pixels within the segmented cell soma (E,i, green outlines) or all pixels within the segmented mitochondria (E,ii, red outlines).
