Annexin 5 cy3

Annexin V-Cy3 (Abcam, Cambridge, United Kingdom) was employed to monitor cellular apoptosis levels after irradiation [51 ]. Cancer cells were loaded with ~30 ug/ml Annexin V-Cy3 in a binding buffer (Abcam, Cambridge, United Kingdom) for 5 min in the dark at 2, 24, and 48 h after irradiation. Annexin V-Cy3 fluorescence was detected via Nikon Eclipse TS 100 microscope (Nikon Corporation, Tokyo, Japan). The imaging setup was as follows: ET excitation, 543 ± 15 nm; ET emission, 610 ± 37.5; objective × 20. The emitted signal was captured and recorded as an image of 1392 × 1040 pixels on a computer monitor using Macro-ImageJ software (National Institutes of Health, Bethesda, MD, USA). Three random areas were selected from each band for apoptosis evaluation of the band. The mean fluorescence intensity of each area was analyzed using Adobe Photoshop CS6 (64 Bit) software (Adobe Systems Inc., San Jose, CA, USA) to determine cell apoptosis levels.

The cells were collected by centrifugation and resuspended in 500 μL of binding buffer and 5 μL of annexin v-Cy3 (BioVision, United States) and incubated at room temperature (RT) for 10 min in the dark. The stained cells were analyzed by flow cytometry using an FL2 channel for annexin v-Cy3.