Understanding the details of the cell cycle at the level of individual cells is critical for both cellular biology and cancer research. While existing methods using specific fluorescent markers have advanced our ability to study the cell cycle in cells that adhere to surfaces, there is a clear gap when it comes to non-adherent cells. In this study, we combine a specialized surface to improve cell attachment, the genetically-encoded FUCCI(CA)2 sensor, an automated image processing and analysis pipeline, and a custom machine-learning algorithm. This combined approach allowed us to precisely measure the duration of different cell cycle phases in non-adherent cells.

Our method provided detailed information from hundreds of cells under different experimental conditions in a fully automated manner. We validated this approach in two different Acute Myeloid Leukemia (AML) cell lines, NB4 and Kasumi-1, which have unique cell cycle characteristics. Additionally, we tested the impact of drugs affecting the cell cycle in NB4 cells. Importantly, our cell cycle analysis system is freely available and has also been validated for use with adherent cells.

In summary, this report introduces a comprehensive, automated method for studying the cell cycle in both adherent and non-adherent cells, offering a valuable tool for cancer research and drug development.

Images were acquired with a Leica Thunder Imager (Leica Microsystems, Wetzlar, Germany), equipped with a Lumencor Spectra X Light Engine (Lumencor, Beaverton, USA) for fluorescence excitation, a motorized stage and a Leica DFC9000 GTC camera. For non-adherent cells, images were acquired with LAS X software (Leica Microsystems, Wetzlar, Germany, version 3.7.5.24914) using a 10X/0.32 NA PH1 dry objective. The mCherry and mVenus signals were detected respectively with 540-580 nm and 460-500 nm excitation filters, 585 and 505 nm dichroic mirrors and 592-668 nm and 512-542 nm emission filters. The brightfield channel was also acquired. Ten fields of views per well were acquired and focal points were manually set in each position before starting the acquisition and kept constant during the whole time-lapse thanks to the Adaptive Focus Control (AFC, Leica Microsystems). The total duration of the time-lapse on non-adherent cells was 120 hours, and the time interval was set to 30 minutes. This dataset represents one position in one well.