A simple technique for quantifying apoptosis in 96-well plates

Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification.

We compared our method and the conventional EB/AO method for quantifying apoptosis of suspension cells (Jurkat) and adherent cells (A375) under normal growth and apoptosis-inducing conditions. We found that our new EB/AO method achieved quantification results comparable to those produced using the conventional EB/AO method for both suspension and adherent cells.