Treatment of primary patient blasts with FLT3 inhibitors. (A) Blasts from patient 7 were cocultured with stroma or grown in suspension culture overnight and then 200nM quizartinib was added to both cultures. Cells were incubated with drug for 48 hours and then stained with propidium iodide and analyzed by flow cytometry. Blasts from patient 7 were cocultured with stroma in the presence and absence of 200nM quizartinib for 14 days, and cells were collected and analyzed by light microscopy (B) and scored for differentiation (C). (D) Cells from panel B were also analyzed for the presence of differentiation markers by flow cytometry as described in “Flow cytometry.” (E) Blasts from patient 7 were cocultured with stroma overnight and then 200nM quizartinib was added. After 1 hour, cells were collected and lysates were analyzed by immunoblotting as described in “Flow cytometry.” (F) Blasts from a 52-year-old man with relapsed, refractory FLT3/ITD AML were cocultured on stroma as in panel B. Shown are cells harvested after 9 days of culture with or without quizartinib.