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The small variation for the largest clusters is a reflection of the fact that they tended to be distributed throughout all, or close to all, of the 15 sections investigated, thus limiting the variation upwards (with a potential that some large clusters are in fact even longer, i.e. larger, regarding sphere number).
Sphere number was determined manually.
Percentage of control sphere number was calculated as (mean sphere number for treated wells/mean sphere number of 0.05% DMSO-treated wells)*100.
Yet the inhibition of ATG4 A led to a decrease in sphere number and size.
Spheres were allowed to form for 14 days at 37 °C and then sphere number was quantified for each well and plotted against the number of cells seeded per well.
Cell numbers were counted, and sphere-forming rate at each time point was calculated as the sphere number of 6 days later divided by cell number of the current time point.
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On Day 6, 12, 18, 24, and 30, sphere numbers were counted and cells were trypsinized into single cells.
The results showed that knockdown of NeuroD1 was associated with a significant reduction in the sphere numbers (Fig. 2a, b).
After culturing for 7 days, sphere numbers were counted under low-power microscope, and the sphere-forming efficiency was expressed as a ratio of spheres to the initial number of single cells.
Mean sphere numbers are presented as Fig 7A and B for clarity, but numbers can be compared as they were seeded simultaneously.
As for primary spheres, the number of secondary spheres was determined under microscope.
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