Annexin 5 pe

A549 and SKOV3 cells were treated with 100 nM 1α,25(OH)₂D₃ for 48 h, following 4 Gy X-ray radiation. After 24 h, the cells were harvested, washed with PBS, and incubated for 15 min at room temperature in a binding buffer containing Annexin V-FITC or Annexin V-PE and PI or 7-AAD (Jiangsu KeyGEN Bio TECH Corp., Ltd, China) before flow cytometry (FC500, Beckman Coulter, Inc., Brea, CA, USA). All the quadrants, except the left lower quadrant, were determined and analyzed by histograms.

Cells were infected with adenovirus for 72 h. Cell cycle and apoptosis were analyzed by flow cytometry. In brief, for apoptotic analysis, cells were stained with AnnexinV-PE and 7-ADD (KeyGenBiotech, China) following manufacturer's instructions. For cell cycle analysis, cells were fixed in 70% pre-cooling ethanol, and incubated with propidium iodide (PI) (Sigma, USA). Cell cycle and apoptosis were analyzed by a Beckman Coulter EPICS Elite flow cytometer. All tests were carried out in triplicates.

An Annexin V-PE(V-phycoerythrin) and 7-AAD (7-amino-actinomycin D) double-staining Apoptosis Detection kit (KeyGEN, Nanjing, China) was used to detect apoptotic activity according to the manufacturer's instructions. Cells were harvested, washed, and incubated for 15 min with annexin V-FITC and PI. The cells were washed and transferred into 12 × 75-mm Falcon 2052 FACS tubes (Becton Dickinson, San Jose, CA), and data from cells were collected on FACS with a Becton Dickinson Biosciences FACScan and CellQuest software version 3.3. This combination allows for the differentiation between early apoptotic cells (annexin V-FITC positive, PI negative), late apoptotic and/or necrotic cells (annexin V and PI positive), and viable cells (unstained).