Cell Cycle Analysis

Mice bearing Med-211FH flank tumors were treated with palbociclib or vehicle for ten days. Tumors were harvested at euthanasia and processed into a single cell suspension. Cells were counted using Vi-Cell (Beckman Coulter) and resuspended in PBS containing 0.5% FBS. Two ml (4×10⁶ cells) was pipetted into eight ml of ice-cold 70% ethanol and incubated at −20°C overnight for fixation and permeabilization. Cells were rehydrated and stained with propidium iodide prior to flow cytometry. Analysis was performed using FlowJo software.