Objective assessment of changes in nuclear morphology and cell distribution following induction of apoptosis

Background: To objectively measure changes in nuclear morphology and cell distribution following induction of apoptosis. Methods: A spontaneously immortalized retinal pigment epithelial cell line (ARPE-19) was cultured for three days in DMEM/F12 with 10% fetal bovine serum followed by 24 hours incubation in staurosporine to induce apoptosis. Cells that were not incubated in staurosporine served as control. Caspase-3 expression in apoptotic cells was demonstrated by quantitative immunofluorescence. Nuclei were counterstained with DAPI. Assessments of nuclear morphology and cell distribution were performed using ImageJ software. Statistical analyses included Student's t-test and Pearson's correlation coefficient. Nearest neighbor analysis was used to assess cell nuclei distribution. Results: Caspase-3 expression in staurosporine-incubated cells increased by 471% +/- 182% compared to control (P = 0.014). Relative to the control, cells in the staurosporine-incubated cultures had smaller average nuclear area (68% +/- 5%; P < 0.001) and nuclear circumference (78 +/- 3%; P < 0.001), while nuclear form factor was larger (110% +/- 1%; P < 0.001). Cell nuclei from the staurosporine-group (R = 1.12 +/- 0.04; P < 0.01) and the control (R = 1.28 +/- 0.03; P < 0.01) were evenly spaced throughout the cultures, thereby demonstrating a non-clustered and non-random cell distribution. However, the staurosporine-incubated group had a significantly lower R-value compared to the control (P = 0.002), which indicated a move towards cell clustering following induction of apoptosis. Caspase-3 expression of each individual cell correlated significantly with the following morphological indicators: circumference of the nucleus divided by form factor (r = -0.475; P < 0.001), nuclear area divided by form factor (r = -0.470; P < 0.001), nuclear circumference (r = -0.469; P < 0.001), nuclear area (r = -0.445; P < 0.001), nuclear form factor (r = 0.410; P < 0.001) and the nuclear area multiplied by form factor) (r = -0.377; P < 0.001). Conclusions: Caspase-3 positive apoptotic cells demonstrate morphological features that can be objectively quantified using freely available ImageJ software. A novel morphological indicator, defined as the nuclear circumference divided by form factor, demonstrated the strongest correlation with caspase-3 expression.