BODIPY-Cyclopamine competition binding assay

HEK293T cells transiently transfected with mSMO were lifted with trypsin (0.25%) and replated at 6×10⁴ cells in 80 μL DMEM + 10%FBS per well in a 96 well tissue-culture treated plate (Falcon, 353219). The plate was incubated at 37°C for 24 hours. Serial dilutions of test compounds (10x final concentrations) were prepared in culture media. 10 μL of diluted compounds or control media were added to the plate after which the plate was incubated at 37°C for 10 min. Next, BODIPY-Cyclopamine was added to each well at the final concentration of 5 nM, except for control wells that were left unstained. Following at least 1.5 hours incubation at 37°C in 5% CO₂, cells were lifted by vigorous pipetting, transferred to conical bottom 96-well plates, and centrifuged at 400 × g for 5 min at 4°C. Supernatant was discarded, cells were re-suspended in 300 μL of PBS + 0.5% BSA (freshly prepared), and the plate was analyzed with Guava benchtop flow cytometer. The results were interpreted with FlowJo software (version v10.1). Dose response curves were constructed in Prism 6 (GraphPad Software, La Jolla, CA).