Facscanto 2 cell cytometer

Cells were grown with doxycycline for 3 days (HN12) or 4 days (JHU-022, NOKs) to induce FPN expression. Cells were harvested by trypsinization and washed with PBS and resuspended in 300 μL of PBS and fixed via drop wise addition of -20°C absolute ethanol to a final concentration of 70% and incubated at -20°C for at least 1 hour. Fixed cells were washed with PBS and resuspended with FxCycle PI/RNase staining solution (ThermoFisher #F10797). Cells were incubated for at least 30 minutes at room temperature. Fluorescence was measured on a BD FACSCanto II cell cytometer using 488nm excitation and emission was collected using a 670LP filter. FlowJo v10.8.1 was utilized to perform cell cycle calculations.

Edu incorporation was measured using the Click-it Plus Edu Alexa Fluor 647 Flow Cytometry Assay Kit (ThermoFisher C10634). Cells were grown for 3 days with doxycycline to induce trans gene expression and were then incubated with 20 μM Edu for 1.5 hours. After Edu incubations, cells were washed and trypsinized. Incorporated Edu was labeled as per the manufacturer’s protocol. After labeling Edu, total nuclear DNA content was stained using FxCycle PI/RNase solution. Fluorescence was measured on a BD FACSCanto II cell cytometer using 633 excitation and emission was collected using a 660nm/20BP filter. PI fluorescence was measured as detailed above. FlowJo v10.8.1 was utilized to perform Edu incorporation calculations.