Microvesicle production during macrophage differentiation. (A) THP-1 cells were treated with vehicle (inset) or alternatively with PMA to induce differentiation and observed by electron microscopy, n = 3. (B) Freshly isolated peripheral blood monocytes were untreated for 30 minutes (inset) or GM-CSF for 4 hours then cultured overnight, n = 6. Images were captured with an AMT camera (Advanced Microscopy Techniques). Representative images are shown in panels A and B, and microvesicles are indicated by the white arrows in both panels. (C) THP-1 cells and peripheral blood monocytes were treated with PMA and GM-CSF, respectively. Microvesicle concentration containing both annexin V positive and negative events from the MV-gated region (supplemental Figure 1A) was quantified by flow cytometry. Shown is the average concentration ± SEM (n = 6). (D) The vitrified microvesicle samples were transferred to a Gatan Cryo holder and visualized with Tecnai G2 F20 ST TEM at 200 kV. Images were captured using a 4k × 4k Gatan Ultrascan CCD camera at a magnification of ×38 000. Representative cryo-TEM images of microvescicles collected using a 16 000g centrifugation from the culture supernatant of PMA-treated THP-1 cells are shown. Large (> 200 nm diameter) and small microvesicles (30-100 nm diameter; inset) are present. Scale bar = 100 nm in the figure and inset.