Separation of neutrophils using annexin V–coated magnetic microspheres shows that PKCδ proteolysis precedes any detectable features of apoptosis. (A) Neutrophils were incubated for 12 hours ex vivo and then separated by using annexin V–coated microspheres, which bind to cells with external exposure of membrane phosphatidylserine, an early marker of apoptosis. Subsequent staining with FITC-conjugated annexin V and flow cytometry shows negative cells in the upper panel and positively selected cells in the lower panel. (B) Lysates prepared from the two fractions were analyzed by immunoblotting with anti-PKCδ antibody. Equal amounts of protein were loaded: lane 1, annexin V negative fraction; lane 2, annexin V positive. This experiment was performed twice with similar results.