Beckman flow cytometry analyzer

Apoptosis assay was conducted using a FITC-Annexin V Apoptosis Detection kit (Bioteke Corporation) according to the manufacturer's protocol. In total, 3x10⁵ cells were plated into a 6-well plate and incubated overnight at 37˚C. Subsequently, the cells were pretreated with fresh DMEM or different concentrations of CDS-3078 (2, 5 and 10 µM) for 24 h at 37˚C. After treatment, the cells were collected and washed three times with ice-cold PBS. The resultant cell pellets were incubated with 5 µl of FITC-conjugated Annexin V and 5 µl of propidium iodide (PI) for 15 min at room temperature. Fluorescence analysis was performed using a Beckman Flow Cytometry Analyzer (Beckman CytoFLEX; Beckman Coulter, Inc.). Cells were classified as early apoptotic (Annexin V-positive/PI-negative), late apoptotic/necrotic (Annexin V-positive/PI-positive), necrotic/dead (Annexin V-negative/PI-positive) and living cells (annexin-negative/PI-negative).
For cell cycle assays, the collected samples were fixed in 70% ice-cold ethanol overnight and washed twice in ice-cold PBS. The resultant cells were stained with in 100 µl PI solution (25 µg/ml RNase A, 50 µg/ml PI) and incubated at 37˚C for 30 min in the dark. The cell cycle distribution was examined using a Beckman Flow Cytometry Analyzer (Beckman CytoFLEX; Beckman Coulter, Inc.) analyzed using Flowjo 10.0.7 software (FlowJo LLC).