Fluorescence image analysis for μSCeNT assay. (A) Representative raw and filtered images from TNF-α dose-response experiments comparing NF-κB activation via TNF-α treatment at 10 ng/mL for 20 minutes to no treatment. (Top to bottom) Raw image of Hoechst stain (nucleus only), and raw image of RelA (NF-κB subunit) stain (whole cell). Scale bar represents 100 μm. Binary cytoplasmic mask generated by subtracting nuclear region from whole cell; inset of cytoplasmic mask; magnified RelA stain with yellow outline of cytoplasmic mask to indicate boundaries separating nucleus and cytoplasm, and color overlay of Hoechst (blue) and RelA (green) stains. Scale bar represents 20 μm. (B) Scatter plots of mean nuclear intensity versus mean cytoplasmic intensity per cell analyzed in a single microchamber. Intensity ratio IR per cell can be calculated, and a histogram can be displayed to show features of the population distribution of IR values (x-axis), where y-axis is relative frequency (R.F.). Gray represents no treatment; blue, TNF-α at 0.1 ng/mL for 20 minutes; and red, TNF-α at 30 ng/mL for 20 minutes. “Replicates” indicates distributions from 9 independent microchambers overlapped.