Figure 2
Patient-derived cells did not respond to extracellular reelin to alter cell motility. Live cells were stained with NucBlue dye to visualize cell nuclei, which was tracked over 24 h to measure cell motility. Age-matched patient cell lines (N=9) were compared with control cell lines (N=9). For each cell line, at least 25 single cells were tracked throughout the whole 24 h duration to generate 25 unique tracks. (a) Representative brightfield image overlaid with NucBlue stained nuclei imaged straight after incubation with NucBlue dye; t=0 h. (b) Harmony software (PerkinElmer, Llantrisant, UK) identified all cell nuclei stained with NucBlue dye based on “Find nuclei” search algorithm; t=0 h. (c) Live cells retain the NucBlue stain after 24 h, imaged as the final frame at the end of the single cell migration assay. (d) Representative image of “Find nuclei” algorithm used to identify all NucBlue stained nuclei at t=24 h. (e) Single cells were tracked for a total duration of 24 h by imaging multiple field of views at one frame per 30 min. Cell motility was quantified by measuring frame-to-frame movement of cell nuclei from “Start” (first imaged frame; t=0 h) to “End” (last imaged frame; t=24 h). (f) Harmony software was used to generate motility tracks of each single cell to represent the accumulated distance covered by each cell over 24 h. (g) Effects of extracellular reelin on olfactory neurosphere-derived cells. Wells were coated with purified reelin or mock-conditioned medium. All assays were run in the same instance in a 96-well plate format. Motility of patient cells (closed gray bars) were directly compared with age-matched healthy control cells (open bars), in the absence (“Mock”) or presence (“Reelin”) of reelin. All plotted data represents mean of all cells measured per group±s.e.m. *P<0.05 based on Tukey’s post-hoc test following two-way ANOVA. Scale bar = 100 μm.
