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David T. Stark, Eric J. Knott, Nicolas G. Bazan; The Morphonuclear Analysis Imaging Method (MAIM): an Unbiased Approach to Quantifying Apoptosis in ARPE-19 Cells Demonstrates NPD1-induced Inhibition of Cell Death. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4106.
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© ARVO (1962-2015); The Authors (2016-present)
Nuclear pyknosis and karyorrhexis are widely used for distinguishing dying cells, but traditional cell death assays based on manual grouping into nuclear morphology categories are time-consuming and prone to observer bias. We introduce here a simple batch image analysis method for assessment of cell death that overcomes these problems through automated quantification of nuclear morphology parameters. We demonstrate the application of this morphonuclear analysis imaging method (MAIM) in ARPE-19 cells.
ARPE-19 cells were cultured on 48-well plates for 72 h to achieve 90% confluence. Cells were fixed in 3.7% neutral buffered formalin, permeabilized for 20 min in 0.1% Triton X-100, and stained with 20 µM Hoechst 33258 in PBS. One 4X4 tile mosaic representing a total growth substrate area of 16.8 mm^2 was acquired from the center of each well using a Zeiss 510 Meta laser confocal microscope and LSM 510 Meta software. Signal was acquired at 8-bit depth, and images were imported into NIH image analysis software ImageJ and batch processed using a custom macro. In brief, an Otsu autothreshold was applied to each image of Hoechst-stained nuclei, and the area of each detected object was recorded. Resulting nuclear size distributions were used to estimate the percentage of pyknotic nuclei in populations of cells exposed to increasing concentrations of staurosporine (STS), a pan-kinase inhibitor and chemical inducer of apoptosis.
Application of MAIM to STS-treated ARPE-19 cells allowed simultaneous assessment of approximately 500,000 nuclei from a single 48-well culture plate and produced characteristic sigmoidal cell death curves. Additional image analysis parameters including nuclear perimeter and mean 8-bit gray value also reflected STS-induced nuclear morphology changes. Neuroprotectin D1 (NPD1), a docosanoid derived from docosahexaenoic acid (DHA; 22:6,n-3) inhibited STS-induced nuclear morphology changes.
Our novel method for batch image analysis of morphonuclear parameters is particularly well-suited to high-throughput analyses of in vitro models of cell death. MAIM is also unbiased, cost-effective, and fully compatible with auxiliary (immuno)cytochemical microscopy techniques. This work was supported by NEI EY005121 (NGB), Research to Prevent Blindness, Inc., and by NIH NS063630 (DTS).
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