*2.8. Flow Cytometry*

For cell cycle studies, cells were trypsinized and fixed for 45 min in methanol/acetone 4:1. After centrifugation at 950 RPM for 10 min, cells were stained with a solution containing 100 μg/mL RNase A and 50 μg/mL propidium iodide (Sigma-Aldrich) overnight in the dark at 4 ◦C. For detection

of HER2 surface expression, cells were trypsinized and fixed for 10 min in 1% paraformaldehyde (Sigma-Aldrich). After blocking with 2% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS, cells were probed with purified mouse anti-human c-erbB-2 (BD Biosciences) overnight in the dark at 4 ◦C. Primary antibody detection was obtained by reaction with secondary antibody Alexa Fluor 488 conjugated with IgG (Thermo Fisher Scientific, # A28175). Flow cytometry analysis was carried out using Fluorescence-activated cell sorting (FACS) Calibur flow cytometer (BD Biosciences). The percentage of cells in each stage of the cell cycle was determined using ModFit software (BD Biosciences). The percentage of cells in Sub-G1 phase was determined using FlowJo X (Tree Star Inc., Ashland, OR, USA).
