Apoptosis measurement by flow cytometry. RAW 264.7 cells were treated with vehicle PBS or DON (1000 ng/ml) for 6 h. Total DON-treated cells and adherent DON-treated cells were collected separately. After washing twice with cold PBS, annexin staining was performed according to the manufacturer's protocol (BD Biosciences). Briefly, cells were resuspended to 13 binding buffer at a concentration of 2.5 3 10 6 cells/ml, and 100 ll of the cell suspension was transferred to fluorescence activated cell sorter tubes. Cells were incubated with 5 ll of annexin V and propidium iodide (PI) for 15 min at room temperature in dark. Finally, samples were resuspended to 400 ll volume with binding buffer, acquired on the Accuri C6 Flow Cytometer (Ann Arbor, MI), and analyzed using FlowJo software (Tree Star, Ashland, OR). Live Raw 264.7 cells were gated using Forward Scatter versus SSC.