Fluorescein conjugated annexin 5 and propidium iodide

K562 and AR230 cells were incubated with the indicated concentrations of bortezomib. The cells were collected by centrifugation, washed with PBS and stained with fluorescein-conjugated annexin V and propidium iodide (BD Biosciences) and the percentage of cells undergoing apoptosis was measured by flow cytometry as previously described [31 (link)].

Cells were seeded overnight at a density of 1×10⁵ in 96-well plates in DMEM containing 10% FBS, and an MTT assay was used to determine the proliferation of different cells exposed to DHA (Sigma, USA). After growing for 48 h, the cells were incubated for 4 h at 37°C with 200 μl MTT solution and measured at 490 nm by using a microplate reader.
For apoptosis assay, the cells were harvested, washed twice with pre-cold PBS, centrifuged at 1000 rpm for 10 min, and resuspended in chilled PBS after growing for 48 h in DMEM containing 10% FBS. Then, the cells were stained with fluorescein-conjugated annexin V and propidium iodide (BD Biosciences) for 15 minutes in the dark at room temperature according to the manufacturer’s instructions for the apoptosis staining, and the percentages of apoptosis cells were analyzed by a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The experimental groups are representative of at least three independent experiments.