In cell developer v1.9.2 analysis software

PANC1 and SNC12 spheres were generated in 1536-well in black, clear bottom microtiter plates (catalog number 3836, Corning) as described above. Once spheres were formed, they were stained with Hoechst dye 33342 (Invitrogen, Carlsbad, CA, USA), at a concentration of 1:1000 and PI (Invitrogen) at a concentration of 1:500 and incubated for 2 h at room temperature. The spheres were then imaged with an IN Cell 2000 (GE Healthcare, Marlborough, MA, USA) using a 10 × 0.45 NA lens. Spheres were imaged with three channels; brightfield, DAPI (350/50 excitation, 455/50 emission), and PI (Texas Red; 579/34 excitation, 624/40 emission) at 50, 100, and 25 ms exposures, respectively. Images were analyzed using GE's IN Cell Developer V1.9.2 analysis software (GE Healthcare). Briefly, the DAPI channel was used to identify two classes of spheroids (large and small) using an intensity segmentation algorithm (objects greater than 255 RFU). Large spheroids were categorized as having an area of 200 μm² or more, whereas small spheroids and individual cells were categorized as having an area of <200 μm². PI objects were also identified with intensity segmentation (>215 RFU) and size criteria (>6 μm²). PI-positive objects in within DAPI spheroids were calculated using standard Developer target linking.