Microvesicle shedding is enhanced in sucrose solution, whereas microvesicle IL-1β loading is facilitated in the presence of high extracellular K⁺. In panel A, mature DCs were stimulated with 60 μM BzATP in high-K⁺ medium. In all other experiments, DCs, whether in high-K⁺ medium, sucrose solution, or standard saline, were stimulated with 200 μM BzATP. Bar = 10 μm. Panel B shows microvesicle protein content (ves) of supernatants from DCs incubated in sucrose solution (□), high-K⁺ medium (▪), or standard saline (⊡). Panel C shows vesicular IL-1β release/10⁶ cells from DCs incubated in the 3 different incubation media (bars as in panel B). In panel D, DCs were incubated in sucrose, high-K⁺ solution, or standard saline solution and stimulated with BzATP. Microvesicles were recovered and IL-1β content of microvesicles (ves) and DCs (DCs) was expressed as pg of cytokine/μg of microvesicle protein or pg of cytokine/μg of cellular protein, respectively (bars as in panel B). Data are averages ± SE of 3 experiments performed with cells from 3 different donors. In panel E, DCs were treated with BzATP in standard saline solution after overnight incubation in the presence or the absence of the irreversible caspase-1 inhibitor Y-VAD–CMK (100 μM). Mature DCs from the same donor were also treated with BzATP in high-K⁺ medium. The purified microvesicles were analyzed by Western blot for the presence of IL-1β. Black arrows in A indicate shed microvesicles.