Apoptosis and necrosis/necroptosis induced distinct morphological features when stained with AO. (a–h) Representative images of 3T3 NIH cells untreated (a and e) or treated with staurosporine (b and f), TNF/zVAD-FMK (c and g), or H₂O₂ (d and h) for 6 (a–d) or 24 (e–h) h. (j, k, and m–p) Morphology can be segmented in AO cells based on nuclear (green AO emission; j, m, and n) and RNA (red AO emissions; k, n, and p) changes. (i–k) Healthy cells had large, well-demarcated granulated nuclei. Staurosporine-induced apoptosis was characterized initially by nuclear shrinkage and cellular fragmentation. Cells later lost their nuclear demarcation, and ultimately lost red AO emission (cellular RNA), exhibiting a fragmented nucleus. (l–p) During necrosis (m and n) or necroptosis (o and p), cells initially had a shrunken nucleus with absent nuclear demarcation. These cells ultimately lacked cellular RNA and possessed a smooth, shrunken, and bright nucleus. Bars: (a–h) 50 µm; (i and l) 10 µm. Data are shown as mean ± SEM.