Celigo s

Cells were prepared for imaging cytometry as described above for flow
cytometry but using half-area, black, clear-bottomed 96-well plates (Greiner
CELLSTAR). Each plate of cells was then centrifuged at 500 x g for 5 minutes at
4°C. Cells were imaged in a Nexcelom Celigo S every 20 minutes for 3
hours at room temperature using the “Target 1+Mask” expression
analysis settings. Target 1 was GFP (green channel) and the brightfield image
was used as a mask to segment cells. Exposure time for GFP was 200,000
μs.
The cells were segmented using Celigo’s native brightfield
algorithm for image analysis. The intensity threshold was set to 10, the
precision was set to high, the cell diameter and dilation radius were set to 4
μm and 0 μm, respectively, and the separate touching objects
setting was turned on. The identified cells were then gated based on the mean
intensity and aspect ratio of mCherry fluorescence to exclude debris and clumps
of cells (Figure 5A). Background correction
was used in the analysis of the GFP intensity. For data analysis and
presentation, the mean GFP intensity was averaged across three to four
biological replicates. Dose-response curves were fitted to a nonlinear Boltzmann
function in GraphPad Prism 4 using a least squares regression.