Figure 4

Purifying cytokinetic cells without chemical labeling.

(A) (Left plot) A histogram showing the bimodal distribution of mAG-Geminin in L1210 cells. Cells expressing a basal mAG-Geminin level (black) are at G1 and late mitosis and can be distinguished from the rest of the population (gray). (Mid plot) A bivariate plot showing mAG-Geminin vs. FSC-W. Large cells (high FSC-W) with low mAG-Geminin levels are framed (red); these are putative cytokinetic cells (Cyt). Large cells (high FSC-W) with high mAG-Geminin levels (gray) are putative G2 and early M cells (G2-M). Small cells (low FSC-W) with low mAG-Geminin (blue) are putative G1 cells. (Right plot) DNA distributions of gated Cyt and G2-M populations are shown in matching colors. The DNA distribution of the entire population is shown in black. (B) L1210 cells were gated and sorted following the mAG-Geminin/FSC-W gating strategy (A, mid plot). The purified cells were fixed and stained with propidium iodide. DNA distributions of Cyt (red), G2-M (gray) and the entire population (black) post-sort are shown. Mean percentage of Cyt cells with 4 N DNA was calculated from 3 independent experiments; SD = 7.58. (C,D) Cytokinetic cells were sorted into PFA-containing dishes and processed for imaging (C) or, alternatively, fixed and then sorted into PBS-containing dishes using the same gating strategy (D). Representative DIC and fluorescent images of sorted cytokinetic cells are shown. Arrowheads indicate cells at early cytokinesis identified by chromatin morphology. Arrows indicate cells at late cytokinesis identified by 8-shaped morphology. In order to simplify chromatin visualization post-sort, cells were pre-labeled with Hoechst 33342 but sorted based on FSC-W and mAG-Geminin signals only. (E–G) Cytokinetic cells were purified by FSC-W and mAG-Geminin (E, red rectangle) without doublet discrimination (marked by the two-cell droplet). The sorted cells were fixed (PFA) and processed for DIC imaging (F). A similar sorting strategy was performed on pre-fixed cells (G). Images of two representative fields are shown (F,G). Percentages of 8-shaped cells post-sort were calculated from 522 (F) and 470 (G) cells. Arrows indicate late cytokinetic cells. Asterisked arrows indicate late cytokinetic cells whose emerging daughter cells appeared at two focal planes. Framed areas in Field 2 were magnified for better visualization.