Fig. 3. Comparison of basophil activation markers according to basophil variables. Indirect flow BAT was performed in positive control sera. The expression percentage for each activation marker (CD203c, DP, and CD63) is plotted according to the four basophil preparation methods (D_NA^UP, D_NA^P, D_A^UP, and D_A^P). To prepare artificial positive control serum, negative control serum was spiked with reconstituted monoclonal anti-FcεRIα antibody. The spiking volume (µL) per 100 µL of negative control serum is indicated by colored markings (see legend). All four types of basophils were tested in parallel by changing the volume of the monoclonal anti-FcεRIα antibody in series of batches. D_A^P basophils, however, were tested only with the minimum volume of 6 µL. Nevertheless, D_A^P basophils displayed the highest SNR and positivity rate (PR, assay sensitivity). Expression percentages of each activation marker were measured by using basophil gate 3.

Abbreviations: FcεRIα, α-chain of IgE receptor; DP, double positive (CD63+CD203c+); SNR, signal to noise ratio; PR, positivity rate, D_NA^UP, non-atopic donor unprimed; D_NA^P, non-atopic donor primed; D_A^UP, atopic donor unprimed; D_A^P, atopic donor primed.