Annexin 5 alexa flour 488 conjugate

Cells were plated and treated in 6 well plates as described above. After 24 h of treatment, 500,000 cell aliquots of each sample were taken, including 4 aliquots of the 10 μM Etoposide positive control and 1 aliquot of each other treatment. Cells were washed twice with cell staining buffer (1% FBS in PBS), and then resuspended in 95 μL 1X Annexin V binding buffer (ThermoFisher) containing Vybrant® Cell Cycle Ruby dye (ThermoFisher) and Brilliant Violet-421™-Annexin V (BioLegend) as per manufacture’s instruction. For combination studies, cells were stained with 1X Annexin V binding buffer (Invitrogen) with 5 μM Sytox® Red dead cell stain (Life Technologies) and Annexin V Alexa Flour™488 conjugate (1:20 dilution) (ThermoFisher). Instrument controls included Sytox® only, Annexin V only, or unstained sample. Samples were then incubated in the dark at room temperature for 15 min and diluted 1:10 in PBS prior to analysis. Flow cytometric analysis was performed at the University of Arizona Translational Flow Cytometry Laboratory on a FACSCanto flow cytometry system (BD Biosciences) or Guava® easyCyte™ flow cytometer (Millipore). Acquired data was analyzed using FlowJo analysis software.