Apoptotic microvesicles derived from 32Dcl3 cells contain modified chromatin. (A) Forward scatter/side scatter (FSC/SSC) plot of microvesicles isolated from apoptotic 32Dcl3 cells, indicating the size of the microvesicle population (gray) according to calibrated nanobeads with the indicated size in μm (black). (B) 32Dcl3-derived apoptotic microvesicles were stained in flow cytometry for lupus mouse-derived mAbs #34, KM-2, BT164 and LG11-2. Histograms for a representative experiment are shown and the isotype control is indicated in each graph (fine line). (C) Mean fluorescence intensity (MFI) of for the indicated mAbs in multiple experiments (n = 10) showing the signal-range (25 to 75%) between different microvesicle preparations. (D) Extracts of 32Dcl3-derived apoptotic microvesicles were probed in western blot with the indicated lupus mouse-derived mAbs to confirm the presence of apoptosis-modified histones. The expected position of the respective histones is indicated on the left side. (E) 32Dcl3-derived apoptotic microvesicles were either directly stained (no fix/perm), or fixed and permeabilized before staining (fix/perm), and show no differences for the signal of LG11-2. FSC/SSC plots of the microvesicle population for both conditions are indicated on the left. On the right a representative histogram is shown for the isotype control (not filled) and mAb LG11-2 (filled) for microvesicles without (black line) or with fixation/permeabilization (gray line). (F) Confocal microscopy of 32Dcl3 cells stained with mAb KM-2. Apoptotic microvesicles still attached to early apoptotic 32Dcl3 cells are indicated (arrowheads). (G) Ultra-structural analysis of isolated 32Dcl3-derived apoptotic microvesicles by electron microscopy. Bar, 3 μm. (H) Median size of isolated microvesicles as determined by measuring the size in multiple electron microscopy pictures.