After 3 washes with 0.25% Triton X-100 PBS cells were incubated with goat anti-rabbit Alexa Fluor 568 for 3 h at 4 C. cells on day 3C5 ( 0.05). There was no significant difference between non-induced and diffuse cells at any time point ( 0.05) (Fig.?1B). The average doubling time for non-induced, diffuse, and inclusion body cells were 1.5, 1.6, and 3.0 d, respectively. Open in a separate window Figure?1. Inclusion body-containing cells have a longer cell cycle than diffuse cells. (A) HttCGFP expression was induced in 14A2.5 cells with 10 M of ponasterone-A for 4 d prior to cell suspension and sorting. Cells with inclusion bodies (population P6) can be sorted based on the GFP signal having a smaller height and width than cells containing diffuse GFP throughout the cell (population P7). (B) Live non-induced, diffuse, and inclusion body cells were sorted prior to plating at 350 cells/well in maintenance medium in a 96-well plate. The PrestoBlue viability assay was used to assess proliferation for 5 d after plating. PrestoBlue is a resazurin-based compound that is converted into a fluorescent product upon reduction by a viable cell, increasing proportionally with cell number. The graph represents the average of 3 independent experiments and error bars indicate the standard error of the mean. Asterisks indicate a significant difference ( 0.05) in proliferation between inclusion body cells and non-induced and diffuse cells by Bonferroni post-test. (C) Non-induced, diffuse, and inclusion body cells were sorted into single cells per well of a 96-well plate. Cells were counted every day for 7 d after plating. The graph represents the average of GANT 58 3 independent experiments, and error bars indicate the standard error of the mean. Asterisk indicates a significant difference ( 0.05) in proliferation between inclusion body cells and non-induced and diffuse cells by Bonferroni post-test. (D) Examples of single-cell proliferation for non-induced (upper), diffuse (middle), and inclusion body cells (lower) (GFP is labeled in green). Proliferation was also measured after each population was sorted into plates, with a single cell per well. Consistent with the population study, diffuse and GANT 58 non-induced cells had significantly greater proliferation than inclusion body cells over 7 d (Fig.?1C, examples in Fig.?1D). Average doubling times were nearly identical when cells were plated 350 cells/well or as single cells per well, with times of 1.5, 1.6, and 2.8 d for non-induced, diffuse, and inclusion body single cells, respectively. A 2-way ANOVA demonstrated a significant interaction between cell population and time GANT 58 ( 0.05), and Bonferroni post-tests CLG4B indicated that all populations had significantly different cell numbers on day 7, with inclusion body cells producing the fewest cells over 7 d ( 0.05). Furthermore, only 7.1 2.7% of the wells with inclusion body cells contained a single cell that divided at least once over a 7-d period, whereas the non-induced and inclusion body cells contained dividing cells in 19.0 3.6% and 17.9 4.7% of the wells, respectively. This may be an artifact due to the longer cell cycle time of inclusion body cells and/or cell death. When induced cells were stained for activated caspase 3, there were nearly double the number GANT 58 of inclusion body cells positive for this indicator of apoptosis (11.1 1.1%) compared with diffuse cells (5.9 0.6%). Therefore, cells containing an inclusion body have reduced proliferation and increased cell death compared with cells containing diffuse Htt. To control for the possibility that cell sorting preferentially changed the growth characteristics of inclusion body cells, proliferation was also assessed upon chemical induction of inclusion bodies in unsorted populations. 2-bromopalmitate (2-BP) reversibly inhibits palmitoylation, which is involved in trafficking Htt to the Golgi and has been shown previously to enhance the formation of inclusion bodies in Htt-expressing cells.17 When exposed to 2-BP during a 2 d induction period, 73.4 2.2% of cells contained inclusion bodies compared to 14.7 2.9% of cells exposed to induction media only. This difference was also evident when cells were analyzed by FACS (Fig.?2A). Analogous to the results of the sorted population growth curve, the.