Analyzing Assay Performance
Data analysis of calcium-flux measurements generally are determined as either the net change in fluorescence signal (typically referred to as DF or max signal-background) or the net change in fluorescence signal over background (DF/Fo or S:B).
The DF/Fo calculation has historically been used for comparing data from different labs (assay development done at one site and HTS done at another site), or with assays performed using different instruments.
Although this method is valid for comparisons between different equipment or lab environments, if a researcher is actually comparing the performance of various calcium indicator reagents on a GPCR of interest using a specific instrument, a more direct measure to use would be DF, which is easily calculated and understood.
Because researchers are most interested in the modulation of GPCR activity, performance analysis should be focused on the response (DF), which is the most direct indication of the second messenger cascade. Furthermore, this measurement doesnt obscure the data analysis by dividing by the baseline fluorescence, which is largely due to extracellular fluorescence and has no relation to the GPCR activity.
If one does use DF/Fo when interpreting results, it may be unclear whether a smaller value is due to smaller response (DF) or higher baseline (Fo). Indicator formulations that do not include a background suppression agent, such as Fluo-4NW Calcium Assay, may yield higher background fluorescence, potentially resulting in lower DF/Fo than assay formulations containing a background suppressor (for example, Calcium-3).
If scientists use the DF/Fo approach in this type of scenario, they may unnecessarily eliminate a high-performance formulation option because the DF/Fo is lower, even though the DF shows the indicator formulation would yield a robust assay (Figure 2).
HTS of GPCRs using calcium indicators is an essential method for drug discovery for many years to come. Newer calcium indicator formulations, such as Fluo-4NW Calcium Assay, present an additional tool that offers improved screening efficiency and reduced risk of nonspecific assay interference. As we continue to develop even more new calcium-indicator formulations for specific receptors, cell types, and experimental requirements, a deeper understanding of the best use of these valuable tools will be critical for navigating the challenges faced in drug discovery.