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and A.T.. high selectivity for cytokinetic cells despite their overall Timonacic low abundance in an asynchronous populace. The sorted cells can then be readily utilized for cell biological, biochemical, and genomic applications to facilitate cytokinesis and cell cycle research. Cell division ends with cytokinesis, a process by which a cell halves its cytoplasm in parallel with chromosome segregation and decondensation to produce two child cells1,2. Balanced cytokinesis is crucial for maintaining genomic integrity and indeed, canonical cytokinesis regulators are often associated with malignancy and other human diseases1,2. Cytokinesis is an intensively analyzed subject in cell biology. Nevertheless, the ability to obtain large quantities of late mitotic or cytokinetic cells remains a challenging bottleneck in the field. Cytokinesis is usually a relatively short process; consequently, the portion of cytokinetic cells within a populace of normally proliferating cells is usually small. In general, this limitation can be overcome by cell cycle blocking brokers that pause cell cycle progression at a specific point via checkpoint mechanisms. However, not every step in the cell cycle can be directly blocked. Focusing on mitosis and cell division, there is a shortage of reagents that induce arrest after sister-chromatid separation. Even if there were such reagents, they would most likely interfere with the process of cytokinesis, thus distorting results and data interpretation. In contrast, pre-metaphase synchronization is usually relatively simple, strong, and inexpensive. Microtubule polymerizing/depolymerizing brokers (e.g., nocodazole and taxol), as Col6a3 well as kinesin inhibitors (e.g., monastrol and S-trityl-L-cysteine), interfere with mitotic spindle assembly3,4,5. Consequently, the metaphase plate cannot be created, the mitotic checkpoint is usually activated, and cells are arrested Timonacic with 4 N DNA and fully condensed chromosomes. This synchronization approach is effective; for example, nocodazole blocks cells at pre-metaphase with nearly 100% efficiency. However, effective synchronization at pre-metaphase requires prolonged exposure to chemicals that are, by definition, hazardous. Synchronization of mammalian cells in cytokinesis (C-phase) is typically achieved by releasing cells from pre-metaphase arrest (observe, for example, Ref. 6). However, pre-metaphase blockers damage cytoskeletal organization, potentially Timonacic introducing unwanted variables to the upcoming cytokinesis. Moreover, cells respond differently to drugs due to i) non-genetic heterogeneity; ii) uneven cell cycle arrest resulting from the random cell cycle position of each cell before treatment; and iii) non-cell autonomous effects. No less heterogeneous is the recovery from drug arrests; for instance, in HEK293 human cells, a substantial proportion of mitotic cells is seen three hours after nocodazole removal despite the short length of mitosis (<1?h)7. Together, these phenomena inevitably limit the quality of synchronization, especially in processes such as cytokinesis that capture a small portion of the mammalian cell cycle. Drug-free synchronization is usually inherently preferable. Biomechanical methods for cell cycle synchronization, including centrifugal elutriation, baby-machine, and size-based sorting7,8,9,10, as well as serum starvation, have proven efficient for synchronization at the G1 phase. However, the cell-to-cell variability in cell cycle progression, also known as dispersion, will significantly reduce synchronization by the time cells reach mitosis7. Therefore, these methods have limited use in the synchronization of cells during cytokinesis. Cell cycle arrest at the G1-S transition (e.g., by double thymidine block) brings cells closer to cytokinesis and does not involve cytoskeletal toxicity. However, any type of cell cycle blocker may dissociate the cell cycle from cell growth in ways that can affect division input10. Furthermore, the combination of heterogeneous response and release from your drug with natural dispersion during S, G2, and early M phases would inevitably lower synchronization during cytokinesis. Therefore, when G1-S synchronization is used to enrich cytokinetic cells, the protocol often involves a second synchronization step in mitosis (observe, for example, Ref. 11). We have recently demonstrated the use of standard circulation cytometry for synchronizing mammalian cells in G1 without blocking cell cycle progression7. Driven by our own need for minimally perturbed late mitotic and cytokinetic cells12,13, we have developed a cytometry-based approach for purifying cytokinetic cells directly from an asynchronous populace of proliferating cells. The method is simple and strong, as exhibited for both adherent and unattached cells. Results Isolating cytokinetic cells by cytometry Fluorescently tagged cell cycle proteins have been widely used as cell cycle markers. These fusion proteins are constitutively expressed, and their level is usually regulated by the ubiquitin-proteasome system, such that the markers temporal proteolysis.