Flow cytometry cell cycle analysis

Cells were plated and grown in 12-well plates overnight to log phase. Cells were pulsed with 25 μM BrdU for 20 min at 37 °C. The cells were washed with PBS × 2 and treated with media containing 5 mM hydroxyurea (Sigma-Aldrich) for 6 h at 37 °C. The HU was removed, the cells were washed with PBS × 2, and fresh media was added to allow recovery for 0, 4, 6, 8, and 12 h. After recovery, the cells were washed with PBS and harvested using 0.25% trypsin. The cells were fixed with 70% ethanol at -20 °C, pelleted at 1000 g, and rehydrated with 2% BSA/0.1% Tween20/PBS. The DNA was denatured with 3 M HCl for 30 min/RT, and the cells were blocked with 2% BSA/0.1% Tween20/PBS for 30 min/RT. BrdU was stained using a rat anti-BrdU antibody (1:200 rat α-BrdU (clone BU1/75; #MCA2060 BioRad)) for 1 h/RT, followed by incubation with 1:500 goat α-rat AlexaFluor-488 (Life Technologies) in the presence of RNAse for 2 h/RT. Equal numbers of cells were stained with 7-AAD (5ug/mL) and analyzed using a Becton Dickinson FACSCalibur flow cytometer and FlowJo software (https://www.flowjo.com/).