Fig 3. Optimization of output readings from different depths of the culture well.

Primary AECs from children with CF were seeded into 96-well culture plates, grown to confluence and transduced with eYFP at MOI 500 for 96 hours. Iodide solution was then injected into each well and eYFP fluorescence then read either from the apical (top) or basal (bottom) location of each well. (A) Analysis revealed that top readings resulted in instabilities of fluorescent signal that coincided with the time of iodide injection. Furthermore, there was also inconsistency in the fluorescence signal readouts independent of whether the cells were transduced or not. (B) In contrast, bottom reading outputs were more stable and consistent, with no instabilities in fluorescent signal intensity. Therefore, all subsequent halide assay measurements were taken from the bottom of the well.