Figure 3.

FloIT detects 2-color inclusions NSC-34 cell lysates. NSC‐34 transiently transfected with TDP-43^WT-tdTomato or TDP-43^G294A-tGFP were either mixed at the end of the incubation (A), or immediately following transfection (B). Alternatively, cells were co-transfected to express both fusion proteins (C). After incubation for either 24, 48 or 72 h cells were lysed and analyzed by FloIT. (A-C) Two-parameter, pseudo-color flow cytometry plots showing identification of inclusions containing TDP-43^WT-tdTomato or TDP-43^G294A-tGFP and inclusions containing both. (D) The total number of TDP-43^WT-tdTomato inclusions including dual color inclusions were enumerated by FloIT and are shown as means ± SEM (n = 3). (E) Confocal microscopy of an NSC-34 cell with an inclusion containing both TDP-43^WT-tdTomato and TDP-43^G294A-tGFP. Dashed line represents cell border from transmission image. Scale bar 5 μm. (F) The percentage of TDP-43 inclusions formed in each treatment at time intervals that contained both TDP-43^WT-tdTomato and TDP-43^G294A-tGFP enumerated by FloIT are shown as means ± SEM (n = 3 independent experiments). Significant differences indicated with p value represent unpaired T-test comparisons between treatments indicated by line.