Cell Cycle Analysis and Relevance for Single‐Cell Gating in Mass Cytometry

Cell cycle analysis by mass cytometry (MC) is hampered by the poor resolution of the Iridium‐labeled DNA intercalator compared to DNA‐specific fluorescent dyes. We report here a minimum cell cycle panel for MC consisting of Ir‐intercalator (DNA content), IdU (S phase), anti‐pS28HistoneH3 (mitosis), anti‐CDT1 (G 1 phase) and anti‐Geminin (non‐G 1 phases). Cell cycle distributions obtained by MC were not significantly different from fluorescence flow cytometry results ( r 2 = 0.98, P < 0.001). Further subdivision of the G 1 and G 2 phases could be done with anti‐pS780RB1 (late G 1 ) and anti‐PLK1 (late G 2 ), respectively. A disadvantage of MC is that aggregates of cells cannot easily be removed while retaining all single cells. We have developed an analysis pipeline including unsupervised clustering by FlowSOM and subsequent single‐cell gating. When performed on cells stained with the cell cycle panel, this analysis pipeline successfully identified debris, dead/apoptotic cells, nonsingle‐cell populations and the major cell cycle phases. The presented cell cycle panel and analysis pipeline thus achieves true single‐cell analysis at the same time as any additional channels in the panel are open for phenotyping and cell cycle‐resolved expression or modification analysis.