Flow cytometric determination of apoptosis by combined annexin V/propidium iodide and by TUNEL assay. (A) HMC-1.1 cells (left panel) and HMC-1.2 cells (right panel) were exposed to bortezomib (100nM, middle panel; 1μM, right panel) or control medium (top panel) at 37°C for 24 hours. Thereafter, cells were washed and incubated with annexin V–fluorescein isothiocyanate. Then, propidium iodide was added. Cells were then washed and analyzed by flow cytometry. (B) Determination of apoptosis by TUNEL assay. HMC-1.1 cells (left panel) and HMC-1.2 cells (right panel) were exposed to bortezomib (100nM, middle panel; 1μM, right panel) or control medium (top panel) at 37°C for 24 hours. Thereafter, a TUNEL assay was performed as described in “Methods.” Cells were analyzed on a Nikon Eclipse E 800 fluorescence microscope (Nikon) equipped with 100×/1.35 UPlan-Apo objective lens (Olympus). Figure acquisition was performed using Olympus DP11 camera and Adobe Photoshop CS2 software Version 9.0 (Adobe Systems). Magnification, ×400.