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R. Hamilton, S. Mukherjee, D. Otteson, A. Giddabasappa CS, J. E. Johnson, S. Chaney, W. Xiao, A. Swaroop, D. G. McMahon, D. A. Fox; A Moderate Throughput Screening (MTS) Flow Cytometric Live/Dead Assay for Retina, Rod Photoreceptors and Dopaminergic Amacrine Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2501.
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© ARVO (1962-2015); The Authors (2016-present)
The aim of this study was to develop and validate a new MTS method for retinal cell counting using the Agilent 2100 Bioanalyzer (AB).
Retinas from different age C57BL/6 mice were isolated and dissociated into a single cell suspension using a gentle and rapid trypsin digestion procedure. The number of live and dead cells was determined using two independent techniques: the standard trypan blue/stereology procedure and calcein-AM (live cells) and ethidium homodimer (dead cells) staining using a modified AB flow cytometric protocol and AB data analysis software. Six mouse models were used: normal adult (60 days old) controls, adult Nrl-GFP transgenics with and without adult lead exposure, adult Nrl-GFP transgenics with and without low-level gestational lead exposure, 5-6 month old Nrl-GFP transgenics, and adult tyrosine hydroxylase (TH)::RFP transgenics.
In normal adult controls, ~2 x 106 cells were isolated per retina. Both analysis protocols revealed that the isolation procedure produced only 3-4% cell death. In adult Nrl-GFP mice, 70-75% of the cells were positive for GFP, which is consistent with the number of mouse retinal rods. Retinas from adult Nrl-GFP mice exposed to a low level of lead for six weeks as adults had ~20% less rods, consistent with the rod-selective apoptosis (Fox et al., 1997). Retinas from 5-6 month old Nrl-GFP mice had ~50% less rods and increased cell death, revealing an age-dependent rod-selective degeneration. Only a few TH::RFP retinal cells were detected, consistent with the small population of retinal dopaminergic amacrine cells (Versaux-Botteri et al., 1984). Retinas from adult Nrl-GFP mice with gestational lead exposure had 25-30% more rods, consistent with the increased number of rod photoreceptors in these mice (Fox and co-workers, ARVO 2006 and 2007).
These results demonstrate that the AB flow cytometry platform can be used as a MTS tool for the determining the effects of drugs, toxicants, fusion proteins, aging or inherited retinal degenerations on different retinal cell populations. The technique is reliable and sensitive enough to detect 1-3 fluorescently-labeled RFP amacrine cells in a population of >1,000 unlabeled cells or 3-4% cell death in control cells. Moreover, it can determine the selective loss or proliferation of rods fluorescently-labeled with Nrl and the occurrence of additional cell death.
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