Fig. 7

ACDC accurately measures cilia length and provides a major speed enhancement in cilia analysis. a htert-RPE1 cells were grown in serum-containing media and then in serum starvation media for 2 days supplemented with or without 100 nM cytochalasin D (CytoD) for the last 8–16 h. Cells were stained against Arl13b and DAPI and images were captured at 60× magnification. Images were acquired on the same day from the same experiment. For both conditions, the ACDC software was used to manually, automatedly, and semi-automatically measure primary cilia length from 25 images. All analysis modes showed that CytoD-treated cilia were significantly longer than cilia that were just serum-starved. There was no significant (n.s.) difference in length between analysis modes within each condition. Cilia count is shown above each data series. For both serum starvation and +CytoD conditions, 10–25 images were used for analysis. b Images of ciliated cells were taken at ×40 magnification (mag.) and then analyzed. Similar mean lengths were observed between ×60-mag. and ×40-mag. serum-starved conditions and also between ×60-mag. and ×40-mag. +CytoD conditions. All analysis modes showed that CytoD-treated cilia were significantly longer than cilia that were just serum-starved. There was no significant (n.s.) difference in length between analysis modes within each condition. Scatter plots are represented as mean length ± standard deviation from one experiment. Statistical significance in which p < 0.001 is represented by (***). Cilia count is shown above each data series. For both serum starvation and +CytoD conditions, 5 images were analyzed by ACDC manual analysis and 190–200 images were analyzed by automated and semi-automated analysis. c, d Cilia analysis rate (number of cilia analyzed/hour) for manual, automated, and semi-automated analyses of the ×60-mag. and ×40-mag. serum condition images from (a) and (b), respectively. Speed enhancement, relative to ACDC manual analysis, is shown at the top of each graph