Flow Cytometry Analysis of Fluorescent BODIPY-Cyclopamine Competition Assay

Fluorescent BODIPY-cyclopamine competition assay was adopted as following. In brief, HEK293 cells were seeded in 6-well plates and transfected with wild type hSmo expression vector. Two days later, cells were seeded in 24-well plates and treated with the compounds. After incubation for 10 h, the cells were harvest and tested by flow cytometry. The green fluorescence of BODIPY-cyclopamine bound to Smo was examined by a flow cytometer (Guava EasyCyte 6HT-2 L, Merck Millipore, Billerica, MA, United States). At least 5000 cells were analyzed for each data point. FACS data were analyzed with InCyte software.