Figure 5. Flow cytometric analysis of the interaction between CL-GUVs and caspase-8-Bid.

(a–b) Short-term effects (20 time points at 20 s intervals, total 6.66 min) of successive additions of procaspase-8 or Bid to GUVs-CL. (a–b) Each product (caspase-8 or Bid) was added progressively, at 1.66-minute intervals, as shown in the recording, and the mean fluorescence of the vesicles was then measured. (a) Caspase-8 (Casp8) was added before Bid whereas, in (b), caspase-8 was added after three successive additions of Bid (10 nM, 40 nM and 60 nM). Even shortly after additions, the enzymatic system was functional, provided that caspase-8 bound to the giant unilamelar liposomes (GUVs). (c) the upper histogram, in black, corresponds to (a), and the lower histogram, in red, corresponds to (b); the occurrence of vesicles with a higher side scatter (SSC), due to procaspase-8/Bid cleavage activity, was recorded and is plotted as a percentage (%) of the total vesicle population. (d) The intensity of Bid-Alexa₆₄₇ fluorescence associated with GUVs is shown as a function of procaspase-8 addition and time, for GUVs with (closed circles) or without (open circles) CL.