Objective assessment of changes in nuclear morphology and cell distribution following induction of apoptosis.
Diagn Pathol
; 9: 92, 2014 May 12.
Article
em En
| MEDLINE
| ID: mdl-24885713
ABSTRACT
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. VIRTUAL SLIDES The virtual slide(s) for this article can be found here http//www.diagnosticpathology.diagnomx.eu/vs/3271993311662947.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Núcleo Celular
/
Apoptose
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Células Epiteliais
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Forma do Núcleo Celular
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Tamanho do Núcleo Celular
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Epitélio Pigmentado da Retina
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article