Validation of morphological criteria for programmed cell death by propidium iodide staining. Putative RP2s were cultured at 8 h-APF (independent experiments with six and four pairs of sister cultures, respectively) in medium with 6 μg/ml 20E (+20E) or no hormone (-20E). After 48 or 72 h, RP2s were independently scored as dead or alive using morphological criteria (MC) or PI staining. (A, B) Photomicrographs show putative RP2s under phase contrast (PC) and epifluorescence optics for GFP and PI (a marker for dead cells). (A) Putative RP2 cultured for 48 h without 20E. The neuron was alive by morphological criteria, exhibited GFP labeling and lacked PI staining. (B) Putative RP2 cultured for 48 h with 20E was dead by morphological criteria, exhibited GFP labeling and had strong nuclear PI staining (arrow). (C) Similar results were obtained by scoring putative RP2s by morphological criteria or PI staining after 48 h (C1) or 72 h (C2) in culture. Both methods showed that significantly fewer putative RP2s survived in cultures with 20E (*P < 0.01, two-tailed Chi square test with Yates continuity correction). Scale bars: 10 μm.