Example of optimization experiment used to determine the digitonin concentration to be used for cytoplasmic extraction of MCF–7. Cells (5 × 10⁴ per well) were extracted for 15 min on a rocking table (lifting frequency 50–60/min) with the indicated digitonin concentrations. The level of plasma membrane permeabilization (LDH activity) and lysosome permeabilization (cathepsin activity) were measured. Cells were completely permeabilized at 200 μg/mL digitonin (total). A digitonin concentration that only permeabilizes the plasma membrane and leaves lysosomes intact is ideal. Here, 15–18 μg/mL is optimal for MCF-7 cells.