Figure 3

‘Doublet indiscrimination’ as a means of isolating late cytokinetic cells.

(A) A schematic showing a stream of two droplets, each occupied by one cell. The bottom drop carries a cell at late cytokinesis. The cell appears as two adjacent cells (doublet) and should be discarded by doublet discrimination. (B) mAG-Geminin-expressing L1210 cells were labeled with Hoechst 33342 and analyzed by FACSAria III. A mAG-Geminin/DNA bivariate plot without doublet discrimination is depicted. Putative cytokinetic cells were gated (red). (C,D) The gated cells were sorted into PFA-containing dishes (C) or, alternatively, fixed and then sorted into PBS-containing dishes (D). Representative DIC and fluorescent images are shown. Arrows indicate late cytokinetic cells, some with a noticeable midbody (see magnified area in D, Field 2). Arrowheads indicate early cytokinetic cells for which condensed chromatin could be observed. Percentages of 8-shaped cells post-sort were calculated from 373 (C) and 390 (D) cells. (E) mAG-Geminin-expressing L1210 cells were stained with Hoechst 33342, fixed (PFA) and immunolabeled with anti-Tubulin and fluorescent secondary antibodies. Cytokinetic immunolabeled cells were processed for sorting into a glass-bottom dish and imaged by a ×100 oil lens. Two representative cells are shown.