Objective assessment of changes in nuclear morphology and cell distribution following induction of apoptosis
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- 作者：Jon R Eidet ; Lara Pasovic ; Rima Maria ; Catherine J Jackson…
- 刊名：Diagnostic Pathology
- 出版年：2014
- 出版时间：December 2014
- 年：2014
- 卷：9
- 期：1
- 全文大小：922 KB
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- 出版者：BioMed Central
- ISSN：1746-1596
文摘
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%?±-82% compared to control (P--.014). Relative to the control, cells in the staurosporine-incubated cultures had smaller average nuclear area (68%?±-%; P--.001) and nuclear circumference (78?±-%; P--.001), while nuclear form factor was larger (110%?±-%; P--.001). Cell nuclei from the staurosporine-group (R--.12?±-.04; P--.01) and the control (R--.28?±-.03; P--.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--.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--.001), nuclear area divided by form factor (r--0.470; P--.001), nuclear circumference (r--0.469; P--.001), nuclear area (r--0.445; P--.001), nuclear form factor (r--.410; P--.001) and the nuclear area multiplied by form factor) (r--0.377; P--.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. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3271993311662947