Surface PDI inhibition affects PS dynamics in ECs. (A) Time-lapse imaging analysis of NBD-PS uptake in ECs. Pretreatment with BD34 anti-PDI (■) delayed the NBD-PS uptake compared with isotype control IgG (◇). The arrow indicates the addition of NBD-PS. Data are mean ± SEM of approximately 20 cells. (B) Flow cytometric analysis of the apparent flippase activity on EA.hy926 cells pretreated with the inhibitory BD34 anti-PDI (■) or isotype control (◇), using NBD-PS and the impermeant quencher sodium dithionite. The internalized PS is represented as the percentile dithionite-resistant NBD-PS fluorescence from total NBD-PS fluorescence. Data are mean ± SEM of more than or equal to 10 000 gated events. (C) Time-lapse imaging analysis of NBD-PS efflux in ECs. Pretreatment of ECs with inhibitory BD34 anti-PDI mAb (■) resulted in accelerated PS efflux and faster dithionite quenching compared with isotype-treated ECs (◇). Data are mean ± SEM of 20 cells corrected for bleaching and normalized against the total NBD-PS fluorescence in the absence of dithionite. (D) Scatter plot representation of NBD-PS efflux in ECs. PDI inhibited (■) or control (◇) ECs were exposed to the impermeant sodium dithionite quencher for 30 minutes, and the PS flop is represented by the difference between the total NBD-PS fluorescence in the absence of dithionite and the residual, dithionite-resistant NBD-PS fluorescence. Statistical analysis was performed by the unpaired t test.