Cellular proliferation assay and immunofluorescence of Ki-67

For cell-proliferation studies, 10,000 to 15,000 cells were seeded in twelve-well plates and treated as described. At each time-point, cells were collected and cell numbers counted using Automated Cell Counter (Countess, ThermoFisher Scientific, Waltham MA). To confirm cell growth data and to relate findings to the standard proliferation marker in the clinical setting, we investigated Ki-67 expression using indirect immunofluorescence. Cells were initially cultured on slides, treated as above, fixed in 4% paraformaldehyde, and permeabilized with 0.5% triton X-100. They were incubated with mouse anti-human Ki-67 antibody (Invitrogen, Carlsbad, CA) followed by goat-anti mouse Alexa-Fluor®488 conjugated secondary antibody (Invitrogen).

Fluorescent sections were then imaged on a Zeiss Axio Observer (Netherlands) and Zeiss Zen software was used for quantification of Ki-67 labeling index (percent of positively-labeled/negatively-labeled cells). An average of four regions was used for calculations.