Real-Time Apoptosis and Necrosis Assay

The RealTime-Glo^™ Annexin V Apoptosis and Necrosis Assay (Promega, Madison, WI, USA) is a live-cell (non-lytic) real-time (kinetic) assay that measures the exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane during the apoptotic process. Measurement of PS exposure is a reliable and well-validated mean of assessing apoptosis [40 (link)]. Briefly, A549 cells were plated at a density of 10,000 cells/well in a white opaque tissue culture 96-well plate (Fisher Scientific, Hampton, NH, USA). The next day, cells were treated with 4X concentration of PFD and PFD–D-Lip, followed by addition of 100 μL of 2X assay buffer and incubated at 37 °C/5% CO₂. Luminescence signal was monitored at regular time points of 0, 3, 6, 12 h using a Tecan microplate reader [41 (link)].