RPE-1 cells expressing H2B-GFP and GFP-NLS were treated as described above to induce micronuclei after depletion of p53 by siRNA. After mitotic shake-off, cells were re-plated and allowed to progress into G1 phase for 4 hours. Afterwards cells were trypsinized and single-cell sorted into 384-well μClear plates (Greiner) using FACS. Following single cell sorting, cells were incubated for 2h to allow for cell attachment and spreading. Plates were mounted on a Nikon TE2000-E2 inverted microscope equipped with the Nikon Perfect Focus system. The microscope was enclosed within a temperature- and CO2-controlled environment that maintained an atmosphere of 37°C and 3-5% humidified CO2. Wells containing single cells of interest were identified manually and fluorescence and DIC images were captured every 30 minutes with a 20X 0.5 NA Plan Fluor objective for up to 48 hours or until the majority of cells had progressed through mitosis. All captured images were analyzed using NIS-Elements software.
Wells containing cells of interest, having completed mitosis, were washed with PBS and cells were subsequently trypsinized. After addition of an excess of fresh medium, daughter cells were separated by limited dilution into new wells in a fresh 384-well μClear plate. Successful separation and transfer into new wells was monitored using a fluorescence microscope. In cases where both daughters ended up in the same well, separation by limited dilution was repeated. After separation, the cells were left to attach for up to 4 hours prior to cell lysis.