Red blood cells naturally can form hemoglobin containing exovesicles. In vitro these vesicle formation is Ca2+ induced. Freshly collected lithium-heparin anticoagulated blood samples were centrifuged for 5 minutes at 400 × g and 4°C. The erythrocytes were washed five times with 20 mM Hepes, pH 7.5, and 150 mM NaCl (washing buffer). A volume of 500 μl washed red blood cells were combined with 3 ml of vesiculation buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM CaCl2 adjusted to 5 μM Ca2+ ionophore A23187) and incubated for 30 minutes at 37°C. After incubation EDTA was added to the solution to a final concentration of 5 mM and centrifuged for 5 minutes at 400 × g and 4°C. The supernatant was collected and centrifuged for 20 minutes at 15,000 × g and 4°C. The resulting pellet was resuspended in washing buffer and centrifuged at 400 × g again to collect a supernatant enriched with red blood cell microvesicles. The supernatant of the 15,000 × g step was further centrifuged for 60 minutes at 100,000 × g and 4°C. The final pellet of this step was resuspended in washing buffer and centrifuged at 15,000 × g again to purify a supernatant enriched in nanovesicles according to [7,20]. The vesicles were assayed for acetylcholine esterase activity and used for further analysis.