Fig. 1. Exposure of oyster hemocytes to 1 µM NA for 24 hours induces membranal changes that are indicative of cells undergoing apoptosis. (A) A scanning electron micrograph of a control hemocyte maintained in vitro for 24 hours in the absence of NA. The cell tends to spread on the substrate (arrowhead) and deploys several pseudopods (arrows), whereas (B) an oyster hemocyte incubated in the presence of 1 µM NA exhibits visible membrane blebbing (arrow). (C) Confocal microscopy analysis after FITC-AnnexinV-PI staining shows that phosphatidylserine is absent from the external leaflet of the plasma membrane, whereas (D) FITC-Annexin V binds to the membrane of hemocytes incubated in the presence of NA, demonstrating that in addition to membrane blebbing (arrow), externalization of phosphatidylserine, one of the earliest events in apoptosis, is occuring. Cells in (C) and (D) were fixed after FITC-Annexin V staining to allow localization of nuclear DNA by PI. Scale bars: (A,B) 1 µm, (C,D) 2 µm.