What does baseline fluorescence level represent for flow assays?

Baseline fluorescence refers to the fluorescence signal that is detected without any amplification, and it is not influenced by the increase in signal associated with the amplification process. This baseline fluorescence primarily arises from two factors: incomplete quenching of the fluorophore in hydrolysis probe assays and the presence of unbound dye in DNA-binding dye assays. In simpler terms, it is the inherent fluorescence exhibited by the sample when there is no specific activity or response taking place. To establish the baseline, the average fluorescence intensity readings before adding the ligand are calculated. Subsequently, when the ligand is introduced, the post-addition fluorescence intensity is divided by this mean baseline signal.