The measurement of multilevel calibrated QuantiBRITE fluorescent beads enables the construction of a standard curve for antigen quantification. Using a Microsoft Excel-spreadsheet referring to CellQuest, provided by BD Biosciences, the sample fluorescence measured can be converted into the term ABC as shown in Fig. 1C for the expression of neprilysin/CD10 on granulocytes. QuantiCALC™ (BD Biosciences) is an automated quantitation analysis software, which automatically converts the FL2 axis to PE molecules per cell and reports statistics in PE molecules per cell. Without these tools a manual analysis can be performed:
1. On a statistics spreadsheet, enter the geometric means from the histogram statistics view for the four beads.
2. Enter the lot specific values for PE molecules per bead from the flyer in the kit.
3. Calculate the log10 for each FL-2 geometric mean and for the PE molecules per bead.
4. Plot a liner regression of log10 PE molecules per bead against log10 fluorescence using the equation y = mx + c.
5. Determine the ABC values for the PE-stained samples by substituting the log FL2 geometric mean of that population and solving for the log ABC (the antilog of this number is the ABC value).