Label Free, Quantitative Single-cell Fate Tracking of Time-lapse Movies

Historically, the ability to perform multi-day time-lapse imaging of adherent cells required expensive and specialized microscopy equipment. As byproduct of this cost, many labs would synchronize cells using inhibitors such as hydroxyurea and thymidine, and or use fluorescent biosensors to minimize time required on the microscope. These methods introduce significant artefacts including phototoxicity, increased DNA replication stress and mitotic defects, thereby limiting the ability to characterize various cell cycle phenotypes. However, increased access to low cost live cell microscopes has removed many of the economic barriers thereby allowing multi-day imaging on asynchronous cells on a regular basis. Here we describe our protocol for manually tracking individual cell fates across multiple generations of random daughter cells using only low toxicity brightfield based imaging. Importantly, our pipeline relies on the free open-source software ImageJ/Fiji and an easy to use Microsoft Excel spreadsheet. Furthermore, annotated files can be saved to allow later recall of any individual cell. In summary, our method provides quantitative data on interphase and mitotic transit time, points of cell cycle arrest and critically, the ability to link these events with cell fate.