Annexin 5 conjugates for apoptosis detection

Human leukemia cells K562 were cultured in RPMI-1640 medium. Cells were cultured with Tig-S (0-20ug/ml) for two days. After drugtreatment, cells were collected by centrifugation, washed with PBS once, and re-suspend in binding buffer (10mM HEPES, 140mM NaCl, 2.5mM CaCl 2 , pH 7.4). Annexin V (Alexa Fluor 488, Invitrogen cat# A13201), and Propidium iodide (1mg/ml in water) were added to cell suspension. The mixture is incubated at room temperature for 15 minutes. Cells were washed with binding buffer 2 times before analysis by flow cytometry. Flow Cytometry was performed in Baylor Core Facility with a LSRII instrument. For reference, control cells were stained with Propidium Iodide (PI) or Annexin V-488 (Annexin V-FITC) alone. Stained cells were analyzed following Invitrogen Annexin V conjugates for Apoptosis Detection protocol. For analysis, cell distribution in following groups: live, early apoptosis, lateapoptosis, total apoptosis and dead cells were determined.